THE    METRIC    SYSTEM 


LIBRARY 

OF  THE 

UNIVERSITY  OF  CALIFORNIA. 


Class 


Q.nan 


190? 


'if. 


\u)  39.3/04  luctics  :  3.2000  ieei  ;    1.0930  yarus. 
Centimeter  (cm.)-io  millimeters  ;  10,000  microns  (//)  o.oi  meter;  0.3937  (f) 

inch. 

Millimeter  (mm. )= i, ooo  microns  (//);  o.icm.;  o.ooi  meter;  0.03937  (^)  inch. 
Micron  (//)  (Unit  of  measure  in  micrometry   (§i82)=o.ooi   millimeter;  one 

millionth  of  a  meter  ;  0.00003937.  (^s^on)  inch. 
Yard;=3  feet ;  36  inches  ;  0.91439  meter  ;  91.4399  centimeters. 
Foot=;[2  inches  ;  30.4799  centimeters  ;  304.799  millimeters. 
Inch=TV  foot  ;  3^  yard;  25.3999  millimeters  (2.54  centimeters). 
later  (Unit  of  capacity)— 1,000  cubic  centimeters  (milliliters);  ( i  quart — . ) 
Cubic  centimeter=o.ooi  liter  (milliliter);  (^  cub.  inch.) 
Fluid  ounce  f  8  fluidrachms)  =29.574  cubic  centimeters  (30  cc. — ). 
Gram  (Unit  of  weight  )=i  cc.  of  water;   15.432  grains. 
Kilogram=  i, ooo  grams  ;  2.2046  (2-i)  Ibs.  avoirdupois. 
Ounce  avoirdupois=437|  grains  ;  28.349  grams. 
Ounce  Troy  or  apothecaries=:48o  grains  ;  31.103  grams 

TEMPERATURE 

To  change  Centigrade  to  Fahrenheit;  (C.  Xf)+32=F.  For  example,  to 
find  the  equivalent  of  10°  Centigrade,  C.  =  io°Xf +32=50°  F. 

To  change  Fahrenheit  to  Cenrigrade  ;  (F. — 32°)Xi~C.  For  example,  to 
reduce  50°  Fahrenheit  to  Centigrade,  (F.=5o°,  and  (50° — 32°)X|=io  C. ;  or 
— 40°  Fahrenheit  to  Centigrade,  F.=  — 40°  ( — 40° — 32°)=  — 72°,  whence  — 
72°Xf=  —40°  C. 


30  grams,  approx. 


Address  of  American  Opticians  :  For  the  price  of  microscopes  and  microscopical  supplies 
the  student  is  advised  to  obtain  a  catalog  of  one  or  more  of  the  opticians.  Nearly  all  of  them 
import  foreign  apparatus.  For  foreign  opticians  see  the  table  of  tube-length  p.  18. 

The  Bausch  &.  l,omb  Optical  Co Rochester,  New  York 

James  T.  Dougherty 409-411  West  sgth  St.,  New  York 

Eimer  &  Amend...' 205-211  3d  Ave.,  New  York 

The  Gundlach-Manhattan  Optical  Company Rochester,  N.  Y. 

E.  Leitz 30  East  iSth  St.,  New  York 

Edward  Pennock 3609  Woodland  Ave.,  Philadelphia,  Pa 

A.  B.  Porter 324  Dearborn  St.,  Chicago 

Queen  &  Company .1010  Chestnut  St.,  Philadelphia.  Pa. 

Spencer  Lens  Company 367-373  Seventh  St.,  Buffalo,  N.  Y. 

Williams.  Brown  &  Earie 918  Chestnut  St.,  Philadelphia.  Pa. 

Voigtlander  und  Sohn,  A.  G 225  Fifth  Ave.,  New  York 

Joseph  Zentmayer 226-228  South  isth  St.,  Philadelphia,  Pa. 

Besides  the  names  here  given,  nearly  every  large  city  has  one  or  more  dealers  in  micro- 
scopes and  microscope  supplies. 


THE    MICROSCOPE 


AN 

INTRODUCTION  TO  MICROSCOPIC 
METHODS    AND    TO    HISTOLOGY 

BY  SIMON  HENRY  GAGE 
Professor  of  Histology  and  Embry- 
ology, Emeritus  in  Cornell  University 


REVISED  AND  ILLUSTRATED  BY  OVER 
TWO  HUNDRED  FIGURES 


COMSTOCK  PUBLISHING  COMPANY 

ITHACA,  NEW  YORK 
1908 


UNIV.   FARM 


Copyright,  1908 

BY  SIMON  HENRY  GAGE 

All  Rights  Reserved 


Printed  by 

Priest  &  Benjamin 

Ithaca,  N.  Y. 


To 

THE  STUDENTS  WHO  HAVE  BEEN 
UNDER  MY  PERSONAL  SUPERVISION, 
AND  TO  THE  UNKNOWN  GROUPS  OF 
WORKERS  WHO  HAVE  RECEIVED  AID 
FROM  EARLIER  EDITIONS,  I  DEDI- 
CATE THIS  TENTH  EDITION,  jt  J* 


185862 


PREFACE  TO  THE  TENTH  EDITION 


WITH  the  general  progress  of  Science,  the  Microscope  and  its  Accessor- 
ies have  not  only  kept  pace,  but  have  added  their  full  share  to  the 
momentum  of   that   progress.     With   the   increased   usefulness  and 
consequent  use  of  the  microscope  it  has  steadily  advanced  in  excellence  and 
efficiency  ;  and  the  means  of  applying  it  to  the  solution  of  the  problems   con- 
fronting the  workers  in  various  fields  are  becoming   more   simple   and  exact 
every  year. 

In  rewriting  this  book  the  aim  has  been  to  represent  the  microscope  of  the 
present  day,  and  to  serve  as  a  helpful  introduction  to  the  microscopic  world. 
Constant  reference  has  been  made  to  original  sources  in  books  and  periodicals 
so  that  the  investigator,  the  teacher  and  the  ambitious  student  might  find 
fuller  treatment  of  any  subject  in  which  he  is  especially  interested. 

Grateful  acknowledgement  is  made  to  the  opticians,  and  the  manufacturers 
of  laboratory  supplies  for  the  loan  of  cuts,  and  for  courteous  and  complete 
answers  to  numerous  questions ;  to  the  directors  of  laboratories  for  helpful 
suggestions;  to  my  colleagues  in  Cornell  University  and  to  my  pupils;  to 
Henry  Phelps  Gage  for  help  in  optics,  micro-chemistry  and  photography  ;  to 
Susannna  Phelps  Gage  for  a  critical  revision  of  the  whole  work,  for  proof 
reading  and  the  preparation  of  the  index.  And  finally  to  Professor  Burt 
Green  Wilder  who  encouraged  me  when  a  student  to  undertake  work  with  the 
microscope,  and  gave  me  every  facility  in  his  power,  I  wish  to  express  special 
feelings  of  gratitude. 

SIMON  HENRY  GAGE, 

CORNELL  UNIVERSITY, 
October  i,  1908  ITHACA,  N.  Y.,  U.  S.  A. 


CONTENTS 


CHAPTER  I 

PAGE 

"The  Microscope  and  its  Parts  :  Lenses  ;  Simple  Microscopes  ;  Com- 
pound Microscopes  ;  Objectives,  Oculars  and  their  Function  ; 
Tube-Length  of  the  Microscope  ;  Field  of  the  Microscope  ; 
Aperture I-  38 

CHAPTER  II 

Lighting  and  Focusing  :  Day  Light  ;  Artificial  Light  ;  Diaphragms  ; 
Condensers  ;  Dark-Ground  Illumination  ;  Ultramicroscopy  ; 
Refraction  and  Color  Images ;  Adjustable  and  Immersion 
Objectives  ;  Testing  a  Microscope  ;  Care  of  the  Microscope 
and  of  the  Eyes;  The  Royal  Microscopical  Society's  Stand- 
ard Sizes  and  Screws;  Laboratory  Microscopes 39-  98 

CHAPTER   III 

Interpretation  of  Appearances   under  the    Microscope  ;  Pedesis  or 

Brownian  Movement  ;  Binocular  Microscopes 99^J!5 

CHAPTER    IV 

Magnification  and  Micrometry  :  Magnification  of  a  Simple  Micro- 
scope ;  Magnification  of  a  Compound  Microscope ;  Varying 
the  Magnification  ;  Camera  Lucida  ;  Micrometry ;  Micro- 
metry with  a  Simple  and  with  a  Compound  Microscope  ;  Unit 
of  Measure  in  Micrometry  ;  Ocular  Micrometers  ;  Filar  Micro- 
meters ;  Varying  the  Ocular  Micrometer  Valuation  ;  Wright's 
Eikonometer 116-140 

CHAPTER  V 

Drawing  with  the    Microscope  :  Camera   Lucida  ;  Magnification   of 

Drawings;  Drawing  with  the  Projection  Microscope 141-154 

CHAPTER  VI 

Micro-Spectroscope  ;  Micro-Polariscope  ;  Micro-Chemistry  ;  Textile 

Fibers;  Food  and  Pharmacological  Products;  Metallography       155-184 

CHAPTER  VII 

The  Abbe  Test  Plate  ;  Apertometer ;  Determination  of  the  Equiva- 
lent Focus  of  Objectives  and  Oculars  ;  Class  Demonstrations  ; 
Individual  Demonstrations  in  Microscopy,  Histology  and 
Embryology • 185-202 


VI  CONTENTS 

CHAPTER  VIII 

Photographing  with  a  Vertical  Camera ;  Photographing  large, 
Transparent  Objects  with  a  Camera  pointed  to  the  Sky  ;  Pho- 
tographing with  a  Microscope  ;  Photographing  Opaque  Objects 
and  the  Surfaces  of  Metals  and  Alloys  ;  Enlargements  ;  Lan- 
tern Slides  and  Bacterial  Cultures 203-244 

CHAPTER    IX 

Slides  and  Cover-Glasses ;  Temporary  and  Permanent  Mounting ; 
Labeling  Microscopic  Slides  ;  Cabinets  and  Trays  for  Storing 
Microscopic  Specimens  ;  Isolation  of  Histologic  Elements  ; 
Preparation  of  Reagents  used  in  Microscopy,  Histology  and 
Embryology 245-183 

CHAPTER  X 

Fixation;  Microtomes  and  Section  Knives ;  Sectioning  Free-Hand, 
with  the  Freezing  Microtome,  with  a  Hand  or  Table  Micro- 
tome ;  Preparation  of  Sections  by  the  Paraffin  Method  ;  by 
the  Collodion  Method  ;  Staining  Microscopic  Preparations  ; 
Mounting;  Serial  Sectioning  of  Embryos  etc.;  Drawings  for 
Book  Illustrations  and  for  the  Preparation  of  Models  ;  Wax 
Models  ;  Models  of  Blotting  Paper 284-332 

Bibliography  _.  333-345 

Index . 346-359 


B 


Parts  of  a  Microscope  :  B  Base  or  foot ;  D  Draw-tube  ;  E  Ocular  or  eye-piece  ;  HA  Handle; 
I  Joint  for  inclination  ;  M  Mirror;  MH  Head  of  the  micrometer  screw  for  fine  adjustment ; 
O  Objective  ;  P  Pillar  ;  PH  Head  of  the  Pinion  for  the  coarse  adjustment  ;  R  Rack  of  coarse 
adjustment  ;  RN  Revolving-  nose-piece  ;  S  Stage  of  the  microscope  ;  SS  Substage  containing 
the  Abbe  condenser ;  T  Body  Tube.  (Cuts  loaned  by  the  Bausch  &  I,om1>  Opt.  Co.) 


The  Microscope  in  Section  with  the  Images:  i.  2,  3,  Beams  of  light  to  the  mirror;  C 
Image  distance,  i.  e.:  from  eye-point,  E-P,  to  virtual  image  (04);  CD  Condenser  diaphragm; 
EP  Eye-point  of  ocular;  Fj  Upper  focal  plane  of  the  objective;  Fa  Lower  focal  plane  of  the 
eye-piece  ;  Iy  Mechanical  tube-length,  /.  e. :  from  top  of  draw-tube  to  screw  for  insertion  of 
objective;  Oj  Object  on  the  stage  ;  62  Image  formed  by  the  objective  were  no  field  lens 
present ;  Os  -.Image  when  field  lens  is  present ;  CU  Virtual  image  ;  A  Optical  tube-length, 
i.  e.:  distance  from  the  upper  focal  plane  of  the  objective  (Fj)  to  lower  focal  plane  of  eye- 
piece F2. 


THE  MICROSCOPE 


AND 


MICROSCOPICAL    METHODS 


CHAPTER  I 


THE  MICROSCOPE  AND  ITS  PARTS 


APPARATUS   AND   MATERIAL   FOR   THIS   CHAPTER 

A  simple  microscope  (3  2,  12);  A  compound  microscope  with  nose-piece 
(Figs.  76-95) ;  eye-shade  (Fig.  67),  achromatic  (I  23),  apochromatic  (\  25) ,  dry 
(%  20),  immersion  (§21),  unadjustable  and  adjustable  objectives  (2  26,  27); 
Huygenian  or  negative  (§  45),  positive  (\  43)  and  compensation  oculars 
(I  46);  stage  micrometer  (Ch.  IV);  homogeneous  immersion  liquid  (§  21); 
mounted  letters  or  figures  (§60);  ground-glass  and  lens  paper  (§  60). 

A  MICROSCOPE* 

§  i.  A  Microscope  is  an  optical  apparatus  with  which  one  may  obtain  a 
clear  image  of  a  near  object,  the  image  being  always  larger  than  the  object  ; 
that  is,  it  enables  the  eye  to  see  an  object  under  a  greatly  increased  visual 
angle,  as  if  the  object  were  brought  very  close  to  the  eye  without  affecting  the 
distinctness  of  vision.  Whenever  the  microscope  is  used  for  observation,  the 
eye  of  the  observer  forms  an  integral  part  of  the  optical  combination  (Figs. 
16,  26). 

§  2.     A  Simple  Microscope. — With  this  an  enlarged,  erect   image  of  an 


*  For  the  History  of  the  Microscope  see  :  Harting,  Poggendorff,  Mayall, 
Carpenter-Dallinger,  Petri ;  and  Gage,  the  Origin  and  Development  of  the 
Projection  Microscope. 


\_CH.  I 


object  may  be  seen.  It  always  consists  of  one  or  more  converging  lenses  or 
lens-systems  (Fig.  16),  and  the  object  must  be  placed  within  the  principal 
focus  (g  12-14).  The  simple  microscope  may  be  held  in  the  hand  or  it  may  be 
mounted  in  some  way  to  facilitate  its  use  (Figs.  19-22). 


FIGS.  1-9,  Showing  the  Principal  Optic  Axis  and  the  Optical  Center  of 
various  forms  of  Lenses. 

Axis.  The  Principal  Optic  Axis,  c-c'.  Centers  of  curvature  of  the  two 
surfaces  of  the  lens.  c.  I.  Optical  center  of  the  lens.  r-rf.  Radii  of  curva- 
turte  of  the  two  lens  snrfaces.  t-t' .  Tangents  in  Fig.  4. 

$3.  Principal  Optic  Axis. — In  spherical  lenses,  i.  e.,  lenses  which  have 
spherical  surfaces,  the  Axis  is  a  line  joining  the  centers  of  curvature  and 
indefinitely  extended.  In  the  figures  (1-9)  this  line  (c-c')  is  broken  except 
where  it  traverses  the  lens.  In  lenses  with  one  plane  surface  (Figs.  3,  6,  7)  the 
radius  of  the  plane  surface  is  any  line  at  right  angles  to  it,  but  in  determining 
the  axis  it  must  be  the  one  which  is  continuous  with  the  radius  of  the  curved 
surface,  consequently  the  axis  in  such  lenses  is  on  the  radius  of  the  curved 
surface  which  meets  the  plane  surface  at  right  angles. 


CH.  7J 


MICROSCOPE  AND  ACCESSORIES 


\  4.  Optical  Center.— The  optical  center  of  a  lens  is  the  point  through 
which  rays  pass  without  angular  deviation,  that  is,  the  emergent  ray  is  parallel 
to  the  incident  ray.  It  is  determined  geometrically  by  drawing  parallel  radii 
of  the  curved  surfaces,  r-r'  in  Figs.  4-9,  and  joining  the  peripheral  ends  of 
the  radii.  The  optical  center  is  the  point  on  the  axis  cut  by  the  line  joining 
the  peripheral  ends  of  the  parallel  radii  of  the  two  lens  surfaces.  In  Figs.  4-5 
it  is  within  the  lens ;  in  6-7  it  is  at  _the  curved  surface,  and  in  the  meniscus 
(8,  9)  it  is  wholly  outside  the  lens,  being  situated  on  the  side  of  the  greater 
curvature. 

In  determining  the  center  in  a  lens  with  a  plane  surface,  the  conditions 
can  be  satisfied  only  by  using  the  radius  of  the  curved  surface  which  is  contin- 
uous with  the  axis  of  the  lens,  then  any  line  at  right  angles  to  the  plane  sur- 
face will  be  parallel  with  it,  and  may  be  considered  part  of  the  radius  of  the 
plane  surface.  (That  is,  a  plane  surface  may  be  considered  part  of  a  sphere 
with  infinite  radius,  hence  any  line  meeting  the  plane  surface  at  right  angles 
may  be  considered  as  the  peripheral  part  of  the  radius.)  In  Figs.  6,  7,  (r')  is 
the  radius  of  the  curved  surface  and  (r)  of  the  plane  surface;  and  the  point 
where  a  line  joining  the  ends  of  these  radii  crosses  the  axis  is  at  the  curved 
surface  in  each  case. 

By  a  study  of  Fig.  4  it  will  be  seen  that  if  tangents  be  drawn  at  the  peri- 
pheral ends  of  the  parallel  radii,  the  tangents  will  also  be  parallel  and  a  ray 
incident  at  one  tangential  point  and  traversing  the  lens  and  emerging  at  the 
other  tangential  point  acts  as  if  traversing,  and  is  practically  traversing  a  piece 
of  glass  which  has  parallel  sides  at  the  point  of  incidence  and  emergence, 
therefore  the  emergent  ray  will  be  parallel  with  the  incident  ray.  This  is  true 
of  all  rays  traversing  the  center  of  the  lens. 

\  5.  Thick  Lenses. — In  all  of  the  diagrams  of  lenses  and  the  course  of 
rays  through  them  in  this  book  the  lenses  are  treated  as  if  they  were  infinitely 
thin.  In  thick  lenses  like  those  figured,  while  there  would  be  no  angular 


FIGS.  10,  ii. — Sectional  vieivs  of 
a  concave  or  diverging  and  a  convex 
or  converging  lens  to  show  that  in  the 
concave  lens  the  principal  focus  is  vir- 
tual as  indicated  by  the  dotted  lines, 
while  with  the  convex  lens  the  focus 
is  real  and  on  the  side  of  the  lens  oppo- 
site to  that  from  which  the  light 
comes. 


10 


ii 


deviation  for  rays  traversing  the  center  of  the  leas,  there  would  be  lateral  dis- 
placement.    This  is  shown  in  Fig.  64  illustrating  the  effect  of  the  cover-glass. 

\  6.     Secondary  Axis. — Every  ray  traversing  the   center    of    the    lens, 
except  the  principal  axis,  is  a  secondary  axis ;  and  every  secondary  axis  is 


MICROSCOPE  AND  ACCESSORIES 


\CH.  / 


more  or  less  oblique  to  the  principal  axis.     In  Fig.  14,  line  (2),  is  a  secondary 
axis,  and  in  Fig.  15,  line  (i).     See  also  Fig.  65. 

I  7.  Principal  Focus. — This  is  the  point  where  rays  parallel  with  the 
axis  and  traversing'the  lens^cross  the  axis  ;  and  the  distance  from  the  focus  to 
the  center  of  the  lens  measured  along  the  axis  is  the  Principal  Focal  Distance. 
In  the  diagrams,  Fig.  10  is  seen  to  be  a  diverging  lens,  and  the  rays  cross  the 
axis  only  by  being  projected  backward.  Such  a  focus  is  said  to  be  virtual,  as 
it  has  no  real  existence.  In  Fig.  n  the  rays  do  cross  the  axis  and  the  focus  is 
said  to  be  real.  If  the  light  came  from  the  opposite  direction  it  would  be 
seen  that  there  is  a  principal  focus  on  the  other  side,  that  is  there  are  two 
principal  foci,  one  on  each  side  of  the  lens.  .These  two  foci  are  both  principal 
foci,  but  they  will  be  equally  distant  from  the  center  of  the  lens  only  when 
the  curvature  of  the  ;two  lens  surfaces  are  equal.  There  may  be  foci  on  sec- 
ondary axes  also,  and  each  focus  on  a  secondary  axis  has  its  conjugate.  In 
the  formation  of  images  the  image  is  the  conjugate  of  the  object  and  con- 
versely the  object  is  the  conjugate  of  the  image. 


FIG.  12. — Double\Convex  Lens,  Showing  Chromatic  Aberration. 

The  ray  of  white  light  (w)  is  represented  as  dividing  into  the  short 
waved,  blue  (b)  and  the\long  waved,  red  (r)  light.  The  blue  (b]  ray  comes  to 
a  focus  nearer  the  lens  and  the  red  ray  (r)  farther  from,  the  lens  than  the 
principal  focus  (f).  Principal  focus  (f)  for  rays  very  near  the  axis  ;  f  and 
f" ,  foci  of  blue  and  red  light  coming  from  near  the  edge  of  the  lens.  The 
intermediate  wave  lengths  would  have  foci  all  the  way  between  f  andf". 

\  8.  Chromatic  Aberration. — This  is  due  to  the  fact  that  ordinary  light 
consists  of  waves  of  varying  length,  and  as  the  effect  of  a  lens  is  to  change  the 
direction  of  the  waves,  it  changes  the  direction  of  the  short  waves  more 
markedly  than  the  long  waves.  Therefore,  the  short  waved,  blue  light  will 
cross  the  axis  sooner  than  the  long  waved,  red  light,  and  there  will  result  a 
superposition  of  colored  images,  none  of  which  are  perfectly  distinct  (Fig.  12). 

|  9.  Spherical  Aberration. — This  is  due  to  the  unequal  turning  of  the 
light  in  different  zones  of  a  lens.  The  edge  of  the  lens  refracts  proportionally 
too  much  and  hence  the^light  will  cross  the  axis  or  come  to  a  focus  nearer  the 
lens  than  a  ray  which  is  nearer  the  middle  of  the  lens.  Thus,  in  Fig.  13,  if 
the  focus  of  parallel  rays  very  near  the  axis  is  at  _/j  rays  (o  i} ,  nearer  the  edge, 
would  come  to  a  focus  nearer  the  lens,  the  focus  of  the  ray  nearest  the  edge 
being  nearest  the  lens. 


CII.  /] 


MICROSCOPE  AND  ACCESSORIES 


FIG.  13.     Double  Convex  Lens,  showing 
Spherical  Aberration. 


i  10.  Correction  of  Chromatic  and  of  Spherical  Aberration. — Every 
simple  lens  has  the  defect  of  both  chromatic  and  spherical  aberration,  and  to 
overcome  this,  kinds  of  glass  of  different  refractive  power  and  different  dis- 
persive power  are  combined,  concave  lenses  neutralizing  the  defects  of  convex 
lenses.  If  the  concave  lens  is  not  sufficiently  strong  to  neutralize  the  aberra- 

FIG.  13.  The  ray  (o)  near 
the  edge  of  the  lens  is 
brought  to  a  focus  nearer 
the  lens  than  the  rav  (i). 
Both  are  brought  to  a  focus 
sooner  than  rays  very  near 
the  a.ris.  (/)  Principal 
focus  for  rays  very  near  the 
axis  ;  (f)  Focus  for  the  ray 
( i) ,  and  (/")  Focus  for  the 
rav  (o).  Intermediate  rays 
would  cross  the  axis  all  the 
way  from  (f  tof). 

tions  of  the  convex  lens,  the  combination  is  said  to  be  under-corrected,  while 
if  it  is  too  strong  and  brings  the  marginal  rays  or  the  blue  rays  to  a  focus 
beyond  the  true  principal  focus,  the  combination  is  over-corrected. 

In  Newton's  time  there  was  supposed  to  be  a  direct  proportion  between 
the  refractive  power  of  any  transparent  medium  and  its  dispersive  power  (i.  e. 
its  power  to  separate  the  light  into  colors).  If  this  were  true  then  the  con- 
tention of  Newton  that  it  would  be  impossible  to  do  away  with  the  color 
without  at  the  same  time  doing  away  with  the  refraction  would  be  true  and 
useful  achromatic  combinations  would  be  impossible.  It  was  found  by  experi- 
ment, however,  that  there  is  not  a  direct  ratio  between  the  refractive  and 
dispersive  powers  for  the  different  colors  in  different  forms  of  glass,  so  that  it 
is  possible  to  do  away  largely  with  chromatic  aberration  and  retain  sufficient 
refraction  to  make  the  combination  serve  for  the  production  of  images.  (See 
also  the  discussion  under  apochromatic  objectives  \  25.) 

Probably  no  higher  technical  skill  is  used  in  any  art  than  is  requisite  in 
the  preparation  of  microscopical  objectives,  oculars  and  illuminators. 

$  ii.  Geometrical  Construction  of  Images. — As  shown  in  Figs.  14-15, 
for  the  determination  of  any  point  of  an  image,  or  the  image  being  known,  to 
determine  the  corresponding  part  of  the  object,  it  is  necessary  to  know  the 
position  of  the  principal  focus  (and  there  is  one  on  each  side  of  the  lens,  \  7), 
and  the  optical  center  of  the  lens  (Figs.  1-9  ).  Then  a  secondary  axis  (2)  in 
Fig.  14,  (i)  in  Fig.  15,  is  drawn  from  the  extremity  of  the  object  and  pro- 
longed indefinitely  above  the  lens,  or  below  it  for  virtual  images.  A  second 
line  ?s  drawn  from  the  extremity  of  the  object,  (3)  in  Fig.  14,  (2)  in  Fig.  15, 
to  the  lens  parallel  with  the  principal  axis.  After  traversing  the  lens  it  must 
be  drawn  through  the  principal  focal  point.  If  now  it  is  prolonged  it  will 
cross  the  secondary  axis  above  the  lens  for  a  real  image  and  below  for  a  virtual 


image.  The  crossing  point  of  these  lines  determines  the  position  of  the  cor- 
responding part  of  the  image.  Commencing  with  any  point  of  the  object  the 
corresponding  point  of  the  image  may  be  determined  as  just  described,  and 
conversely  commencing  with  the  image,  corresponding  points  of  the  object 
may  be  determined. 

FIGS.  14  AND  15.  14.  Convex 
lens  showing  the  position  of  the 
object  (A-B)  outside  the  principal 
focus  (F),  and  the  course  of  the 
rays  in  the  formation  of  real 
images.  To  avoid  confusion  the 
rays  are  drawn  from  only  one 
point. 

A  B.  Object  outside  the  prin- 
cipal focus.  B'  A'.  Real,  en- 
larged image  on  the  opposite  side 
of  the  lens. 

Axis.  Principal  optic  axis, 
f,  2,3.  Rays  after  traversing  the 
lens.  They  are  converging,  and 
consequently  form  a  real  image. 
The  dotted  line  and  the  line  (2) 
give  the  direction  of  the  rays  as  if 
unaffected  by  the  lens.  (F).  The 
principal  focus. 

FIG.  15.  Convex  lens,  show- 
ing th'e  position  of  the  object  (A  B) 
within  the  principal  focus  and  the 
course  of  the  rays  in  the  formation 
of  a  virtual  image. 

A  B.  The  object  placed  between  the  lens  and  its  focus  ;  A'  B'  virtual 
image  formed  by  tracing  the  rays  backward.  It  appears  on  the  same  side  of 
the  lens  as  the  object,  and  is  erect  ($  //). 

Axis.     The  principal  optic  axis  of  the  lens.     F.    The  principal  focus. 

i,  2,  j.  Rays  from  the  point  B  of  the  object.  They  are  diverging  after 
traversing  the  lens,  but  not  so  divergent  as  if  no  lens  were  present,  as  is  shown 
by  the  dotted  lines.  Ray  (/)  traverses  the  center  of  the  lens,  and  is  therefore 
not  deflected.  It  is  a  secondary  axis  ($6). 


SIMPLE   MICROSCOPE  :    EXPERIMENTS 


§  12.  Employ  a  tripod  or  other  simple  microscope,  and  for 
object  a  printed  page.  Hold  the  eye  about  two  centimeters  from 
the  upper  surface  of  the  magnifier,  then  alternately  raise  and  lower 


CH.  /] 


MICROSCOPE  AND  ACCESSORIES 


the  magnifier  until  a  clear  image  may  be  seen.  (This  mutual 
arrangement  of  microscope  and  object  so  that  a  clear  image  is  seen, 
is  called  focusing.)  When  a  clear  image  is  seen,  note  that  the  let- 
ters appear  as  with  the  unaided  eye  except  that  they  are  larger,  and 
the  letters  appear  erect  or  right  side  up,  instead  of  being  inverted,  as 
with  the  compound  microscope  (§  15,  Fig.  15). 


FIG.  16.  Diagram  of  the  simple  micro- 
scope showing  the  course  of  the  rays  and  all 
the  images,  and  that  the  eye  forms  an  integral 
part  of  it. 

A1  -5'.  The  object  within  the  principal 
focus.  A^  B^.  The  virtual  image  on  the  same 
side  of  the  lens  as  the  object.  It  is  indicated 
with  dotted  lines,  as  it  has  no  actual  existence. 

£2  A2.  Retinal  image  of  the  object  (A^JS1). 
The  virtual  image  is  simply  a  projection  of  the 
retinal  image  in  the  field  of  vision. 

.  I. vis.  The  principal  optic  axis  of  the 
microscope  and  of  the  eye.  Cr.  Cornea  of  the 
eye.  L.  Crystalline  lens  of  the  eye.  R.  Ideal 
refracting  surface  at  which  all  the  refractions 
of  the  eye  may  be  assumed  to  take  place. 


§  13.  Obtaining  the  Principal  Focus. — Hold  the  simple 
microscope  directly  toward  the  sun  and  move  it  away  from  and 
toward  a  piece  of  printed  paper  until  the  smallest  bright  point  is 
obtained.  This  is  the  burning  point  or  focus  and  as  the  rays  of  the 
sun  are  nearly  parallel,  the  burning  point  represents  approximately 
the  principal  focus  (Fig.  1 1 ) .  The  above  and  following  operations 
are  more  easily  accomplished  if  the  lens  is  supported  as  in  Fig.  22. 

§  14.  Real  and  Virtual  Images  with  a  Simple  Micro- 
scope.— Without  changing  the  position  of  the  magnifier  or  paper 
look  into  the  magnifier,  holding  the  eye  close  to  the  upper  surface 
and  the  letters  on  the  paper  may  be  seen,  but  they  will  appear  much 
sharper  to  the  eyes  of  most  people  if  the  magnifier  is  brought  nearer 
to  the  paper,  that  is  so  that  the  printed  paper  is  within  the  principal 
focal  distance  (Fig.  15  and  16). 

After  getting  as  clear  an  image  as  possible  by  focusing  the 
simple  microscope,  raise  the  magnifier  until  the  letters  are  at  a  dis- 


MICROSCOPE  AND  ACCESSORIES 


.  / 


tance  a  little  greater  than  the  principal  focal  distance.  L,ook  into 
the  magnifier  and  note  the  clearness  of  the  virtual  image,  then 
slowly  elevate  the  head  above  the  magnifier  and  when  the  eye  is 
about  60  to  100  centimeters  above  the  lens  a  real  image  can  be  seen. 
That  is  an  image  in  which  the  letters  are  inverted  as  with  the 
objective  of  the  compound  microscope  (see  §  60).  If  the  magnifier 
is  raised  somewhat  so  that  the  printed  letters  are  markedly  without 
the  principal  focus  the  real  image  will  be  seen  more  clearly  espec- 
ially if  the  eye  is  brought  somewhat  near  the  magnifier.  The  above 
experiments  show  two  things. 


FIG.  17.  Figures  of  a  normal  (enime- 
tropic),  a  far  sighted  (hyperopic}  and  a 
short  sighted  (myopic)  eye  to  show  that 
when  the  eye  is  at  rest  the  normal  eye  (E) 
focuses  parallel  rays  on  the  retina  while  the 
far-sighted  eye  (H)  focuses  parallel  rays 
beyond  the  retina.  The  short  sighted  eye 
(M)  focuses  parallel  rays  in  front  of  the  re- 
tina. The  dotted  lines  show  that  in  the 
hyperopic  eye  the  rays  must  be  converging 
to  come  to  a  focus  on  the  retina  zchile  with 
the  myopic  eye  they  must  be  diverging. 


(1)  That  every  convex  or  converging  lens  or  lens  system  can 
serve  to  form  either  a  virtual  or  a  real  image,  depending  upon  its 
position  with  reference  to  the  object. 

(2)  They  show  also  that  without  changing  the  position  of  the 
magnifier,   if  it  is  slightly  further  from  the  object  than  its  principal 
focal  distance,  either  a  virtual  image  or  a  real  image  may  be  seen  by 
many  people,   depending  upon  the  position  of  the  eye.      (a)   If  the 
eye  >is  close  to  the  magnifier  an  enlarged  erect  virtual  image  will  be 
seen,      (b)   With  the  eye  at  a  considerable   distance   an    enlarged 
inverted  real  image  may  be  seen. 

While  the  law  is  absolute  that  real  images  are  formed  only 
when  the  object  is  without  the  principal  focal  distance,  and  virtual 
images  only  when  the  object  is  within  the  focus,  the  above  experi- 
ments show  most  conclusively  that  the  eye  is  a  part  of  the  optical 


CH.  /] 


MICROSCOPE  AND  ACCESSORIES 


arrangement  when  the  microscope  is  actually  used  for  observation, 
and  that  the  microscope  with  the  eye  is  a  different  apparatus  from 
the  microscope  considered  by  itself. 

FlG.  18.  Figure  to  show  that  with 
a  simple  microscope  if  the  object  is  slight- 
ly beyond  the  principal  focus  (F)a  real 
image  will  be  formed  at  A'  which  can  be 
seen  by  an  eye  at  E,  and  thai  if  a  normal 
or  hyperopiceye  is  at  E'  a  virtual  image 
can  be  seen  without  changing  the  posi- 
tion of  the  simple  microscope.  The  long- 
sighted eye  can  see  this  image  best  as  it 
naturally  focuses  converging  rays  on  the 
retina.  The  myopic  eye  either  sees  no 
image  at  all,  or  a  mere  blur,  depending 
upon  the  amount  of  myopia.  A.  object  ; 
A/  real  image  above  the  magnifier; 
A."  virtual  image  which  can  be  seen 
below  the  lens  by  an  eye  at  E' ;  E.  eye  in 
position  to  see  a  real  image ;  E.'  eye  in 
position  to  see  A"  a  virtual  image  ;  F. 
principal  focus  of  the  magnifier. 


A"  < 


FIG.  fo.     Tripod  Magnifier 


The  diagrams,  Figs.  17,  18,  are  introduced  to  show  under  what 
conditions  both  a  virtual  and  a  real  image  may  be  seen  without 
changing  the  position  of  the  magnifier  or  the  object. 

Simple  microscopes  are  very  convenient  when  only  a  small 
magnification  (Ch.  IV)  is  desired,  as  for  dissecting.  Achromatic 
triplets  are  excellent  and  convenient  for  the  pocket.  For  use  in 
conjunction  with  a  compound  microscope,  the  tripod  magnifier  (Fig. 
19)  is  one  of  the  best  forms.  For  many  purposes  a  special  mechan- 
ical mounting  is  to  be  preferred. 


MICROSCOPE  AND  ACCESSORIES  [CH.  I 


FIG.  20      Lens-holder.  FIG.  21.     The  Hastings  Triplet. 


FIG.  22.     Dissecting  Microscope. 


CH.J] 


MICROSCOPE  AND  ACCESSORIES 


FIGS.  23,  24,  25.  Diagrams  showing  the  formation  of  real  and  of  virtual 
images  and  of  the  retinal  image  in  using  the  simple  microscope.  See  the 
explanation  of  Figs.  14,  75,  16. 

COMPOUND  MICROSCOPE 

\  15.  A  Compound  Microscope. — This  enables  one  to  see  an  enlarged, 
inverted  image.  It  always  consists  of  two  optical  parts — an  objective,  to  pro- 
duce an  enlarged,  inverted,  real  image  of  the  object,  and  an  ocular  acting  in 
general  like  a  simple  microscope  to  magnify  this  real  image  (Fig.  26).  There 
is  also  usually  present  a  mirror,  or  both  a  mirror  and  some  form  of  condenser  or 
illuminator  for  lighting  the  object.  The  stand  of  the  microscope  consists  of 
certain  mechanical  arrangements  for  holding  the  optical  parts  and  for  the 
more  satisfactory  use  of  them.  (See  frontispiece.) 

§  16.  The  Mechanical  Parts  of  a  laboratory,  compound  microscope  are 
shown  in  the  frontispiece,  and  are  described  in  the  explanation  of  that  figure. 
The  student  should  study  the  figure  with  a  microscope  before  him  and  become 
thoroughly  familiar  with  the  names  of  all  the  parts. 


OPTICAL   PARTS 

\  17.  Microscopic  Objective. — This  consists  of  a  converging  lens 
or  of  one  or  more  converging  lens-systems,  which  give  an  enlarged,  inverted, 
real  image  of  the  object  (Figs.  14,  26).  And  as  for  the  formation  of  real 
images  in  all  cases,  the  object  must  be  placed  outside  the  principal  focus,  in- 
stead of  within  it,  as  for  the  simple  microscope.  (See  $\  12,  60,  Figs.  16,26.) 

Modern  microscopic  objectives  usually  consist  of  two  or  more  systems  or 


MICROSCOPE  AND  ACCESSORIES 


[CH.  I 


combinations  of  lenses,  the  one  next  the  object  being  called  ih&  front  combina- 
tion or  lens,  the  one  farthest  from  the  object  and  nearest  the  ocular,  the  back 
combination  or  system.     There  may  be  also  one  or  more  intermediate  sys- 
tems.    Each   combination    is,    in 

2  general,  composed  of  a  convex  and 

a  concave  lens.  The  combined  ac- 
tion of  the  system  serves  to  pro- 
duce an  image  free  from  color  and 
from  spherical  distortion.  In  the 
ordinary  achromatic  objectives  of 
the  older  period  the  convex  lenses 
are  of  crown  and  the  concave 
lenses  of  flint  glass.  In  the  best 
modern  achromatic  objectives  the 
new  Jena  glass  is  used  for  a 
part  or  all  of  the  lenses.  (Figs. 
27,  28.) 

FIG.  26.  Diagram  showing 
the  principle  of  a  compound  micro- 
scope with  the  course  of  the  rays 
from  the  object  (AB)  through  the 
objective  to  the  real  image  (B'  A'} , 
thence  through  the  ocular  and  into 
the  eye  to  the  retinal  image  (A*B*), 
and  the  projection  of  the  retinal 
image  into  the  field  of  vision  as 
the  virtual  image  (B^Ai) . 

A  B.  The  object.  A*B\  The 
retinal  image  of  the  inverted  real 
image )  (B* A*},  formed  by  the  ob- 
jective. B^A*.  The  inverted  vir- 
tual image,  a  projection  of  the 
retinal  image. 

Axis.  The  principal  optic 
axis  of  the  microscope  and  of  the 
eye. 

Cr.  Cornea  of  the  eye.  L. 
Crystalline  lens  of  the  eye.  R. 
Single,  ideal,  refracting  surface 
at  which  all  the  refractions  of 
the  eye  may  be  assumed  to  take 
place. 

F.  F.  The  principal  focus  of 
the  positive  ocular  and  of  the  ob- 
jective. 


CH.  I] 


MICROSCOPE  AND  ACCESSORIES 


Mirror.  The  mirror  reflecting  parallel  rays  to  the  object.  The  light  is 
central.  See  Ch.  II. 

Pos.  Ocular.  An  ocular  in  which  the  real  image  is  formed  outside  the 
ocular.  Compare  the  positive  ocular  with  the  simple  microscope  (Fig.  16). 


NOMENCLATURE    OR    TERMINOLOGY    OF   OBJECTIVES 

\  18.  Equivalent  Focus. — In  America,  England,  and  now  also  on  the 
Continent,  objectives  are  designated  by  their  equivalent  focal  length.  This 
length  is  given  either  in  inches  (usually  contracted  to  in.)  or  in  millimeters 
(mm.)  Thus:  An  objective  designated  r'2  in.  or  2  mm.,  indicates  that  the 
objective  produces  a  real  image  of  the  same  size  as  is  produced  by  a  simple 
converging  lens  whose  principal  focal  distance  is  T'2  inch  or  2  millimeters 
(Fig.  n ).  An  objective  marked  3  in.  or  75  mm.,  produces  approximately  the 
same  sized  real  image  as  a  simple  converging  lens  of  3  inches  or  75  millimeters 
focal  length.  And  in  accordance  with  the  law  that  the  relative  size  of  object 
and  image  vary  directly  as  their  distance  from  the  center  of  the  lens  (Figs.  14, 
15,  see  Ch.  IV,)  it  follows  that  the  less  the  focal  distance  of  the  simple  lens  or 
of  the  equivalent  focal  distance  of  the  objective,  the  greater  is  the  size  of  the 
real  image,  as  the  tube-length  remains  constant  and  the  image  in  all  cases  is 
formed  at  160  or  250  mm.  from  the  objective. 

2  19.  Numbering  or  Lettering  Objectives. — Instead  of  designating 
objectives  by  their  equivalent  focus,  many  Continental  opticians  use  letters  or 
figures  for  this  purpose  ;  in  most  cases,  however,  the  equivalent  focus  is  also 

FIG.  27.  Section  of  a  dry  objective 
showing  working  distance  and  lighting  by 
"reflected  light. 

Axis.  The  principal  optic  axis  of  the 
objective. 

B  C.  Back  Combination,  composed  of 
a  plano-concave  lens  of  flint  glass  (F),  and 
a  double  convex  lens  of  crown  glass  (c). 

F  C.     Front  Combination. 

C,  O,  si.  The  cover-glass,  object  and 
slide. 

Mirror.  The  mirror  is  represented  as 
above  the  stage,  and  as  reflecting  parallel 
rays  from  its  plane  face  upon  the  object. 

Stage.  Section  of  the  stage  of  the  mi- 
croscope. 

W.  The  Working  Distance,  that  is  the  distance  from  the  front  of  the 
objective  to  the  objtct  ivhen  the  objective  is  in  focus. 

given.  With  this  method  the  smaller  the  number,  or  the  earlier  in  the  alpha- 
bet the  letter,  the  lower  is  the  power  of  the  objective.  (See  further  in  Ch.  IV, 
for  the  power  or  magnification  of  objectives.)  This  method  is  entirely  arbi- 


MICROSCOPE  AND  ACCESSORIES 


\CH.  I 


trary  and  does  not,  like  the  one  above,  give  direct   information  concerning 
the  objective. 

§  20.  Air  or  Dry  Objectives. — These  are  objectives  in  which  the  space 
between  the  front  of  the  objective  and  the  object  or  cover-glass  is  filled  with 
air  (Fig.  27).  Most  objectives  of  low  and  medium  power  (i.  e.t  \  in.  or  3  mm. 
and  lower  powers)  are  dry. 

§21.  Immersion  Objectives. — An  immersion  objective  is  one  with  which 
there  is  some  liquid  placed  between  the  front  of  the  objective  and  the  object  or 
cover-glass.  The  most  common  immersion  objectives  are  those  (A)  in  which 
water  is  used  as  the  immersion  fluid,  and  ( B)  where  some  liquid  is  used  having 
the  same  refractive  and  dispersive  power  as  the  front  lens  of  the  objective. 
Such  a  liquid  is  called  homogeneous,  as  it  is  optically  homogeneous  with  the 
front  glass  of  the  objective.  It  may  consist  of  thickened  cedar  wood  oil  or 
glycerin  containing  some  salt,  as  stannous  chlorid  in  solution.  When  oil  is 
used  as  the  immersion  fluid  the  objectives  are  frequently  called  oil  immersion 
objectives.  The  disturbing  effect  of  the  cover-glass  (Fig.  64)  is  almost  wholly 
eliminated  by  the  use  of  homogeneous  immersion  objectives,  as  the  rays 
undergo  very  little  or  no  refraction  on  passing  from  the  cover-glass  through  the 
immersion  medium  and  into  the  objective  ;  and  when  the  object  is  mounted 
in  balsam  there  is  practically  no  refraction  in  the  ray  from  the  time  it  leaves 
the  balsam  till  it  enters  the  objective. 

FIG.  28.  Sectional  view  of  an  Immersion, 
Adjustable  Objective,  and  the  object  lighted 
with  axial  or  central  and  with  oblique  light. 

Axis.  The  principal  optic  axis  of  the 
objective. 

B  C,  M  C,  F  C.  The  back,  middle  andt 
front  combination  of  the  objective.  In  this 
case  the  front  is  not  a  combination,  but  a 
single  plano-convex  lens. 

A,  B.  Parallel  rays  reflected  by  the  mir- 
ror axially  or  centrally  upon  the  object. 

C.     Ray  rejlected  to  the  object  obliquely. 
I.     Immersion  fluid  beticeen  the  front  of 
the  objective  and  the  cover  glass  or  object  (O). 
Mirror.     The  mirror  of  the  microscope. 
O.     Object.     It  is  represented   without  a 
cover-glass.      Ordinarily  objects  are    covered 
whether  examined  with  immersion  or  with  dry 
objectives. 

Stage.  Section  of  the  stage  of  the  micro- 
scope. 

$  22.  Non-Achromatic  or  Chromatic  Objectives. — These  are  objectives 
in  which  the  chromatic  aberration  is  not  corrected,  and  the  image  produced  is 
bordered  by  colored  fringes.  They  show  also  spherical  aberration  and  are 
used  only  on  very  cheap  microscopes.  (|$  8,  9,  Figs.  12,  13. ) 


CH.  /]  MICROSCOPE  AND  ACCESSORIES  15 

£  23.  Achromatic  Objectives. — In  these  the  chromatic  and  the  spherical 
aberration  are  both  largely  eliminated  by  combining  concave  and  convex 
lenses  of  different  kinds  of  i*lass  "so  disposed  that  their  opposite  aberrations 
shall  correct  each  other."  All  the  better  forms  of  objectives  are  achromatic 
and  also  aplanatic.  That  is,  enough  of  the  various  spectral  colors  come  ap- 
proximately to  the  same  focus  to  give  white  light.  (See  also  under apochro- 
matics,  \  25.) 

|  24.  Aplanatic  Objectives,  etc. — These  are  objectives  or  other  pieces  of 
optical  apparatus  (oculars,  illuminators,  etc.),  in  which  the  spherical  distor- 
tion is  wholly  or  nearly  eliminated,  and  the  curvatures  are  so  made  that  the 
central  and  marginal  parts  of  the  objective  focus  rays  at  the  same  point  or 
level.  Such  pieces  of  apparatus  are  usually  achromatic  also. 

£  25.  Apochromatic  Objectives. — A  term  used  by  Abbe  to  designate  a 
form  of  objective  made  by  combining  new  kinds  of  glass  with  a  natural  min- 
eral (Calcium  fluorid,  Fluorite,  or  Fluor  spar  1886*).  The  name,-  Apochro- 
matic, is  used  to  indicate  the  higher  kind  of  achromatism  in  which  rays  of 
three  spectral  colors  are  combined  at  one  focus,  instead  of  rays  of  two  colors 
as  in  the  ordinary  achromatic  objectives.  Some  of  the  early  apochro- 
matics  deteriorated  rather  quickly  in  hot  moist  climates.  Those  now  made 
are  quite  permanent. 

The  special  characteristics  of  these  objectives,  when  used  with  the  "com- 
pensating oculars"  are  as  follows  : 

1 i )  Three  rays  of  different  color  are  brought  to  one   focus,   leaving  a 
small  tertiary  spectrum  only,  while  with  objectives  as  formerly  made  from 
crown  and  flint  glass,  only  two  different  colors  could  be  brought  to  the  same 
focus. 

(2)  In  these  objectives  the  correction  of  the  spherical  aberration  is  ob- 
tained for  tzi'o  different  colors  in  the  brightest  part  of  the  spectrum,  and   the 
objective  shows  the  same  degree  of  chromatic  correction  for  the  marginal  as 
for  the  central  part  of  the  aperture.       In  the  old  objectives,  correction  of  the 
spherical  aberration  was  confined  to  rays  of  one  color,  the  correction  being 
made  for  the  central  part  of  the  spectrum,  the  objective  remaining  under-cor- 
rected spherically  for  the  red  rays  and  0t'<?r-corrected  for  the  blue  rays  (§  10). 

(3)  The  optical  and  chemical  foci  are  identical,  and  the  image  formed 
by  the  chemical  rays  is  much  more  perfect  than  with  the  old  objectives,  hence 
the  new  objectives  are  well  adapted  to  photography. 

(4)  These  objectives  admit  of  the  use  of  very  high  oculars,  and  seem  to 
be  a  considerable  improvement  over  those  made  in  the  old  way  with  crown 
and  flint  glass.     According  to  Dippel  (Z.  w.  M.  1886,  p.  300)  dry  apochromatic 
objectives  give  as  clear  images  as  the  same  power  water  immersion  objectives 
of  the  old  form. 


*According  to  F.  J.  Keeley  (Proc.  Acad.  Nat.  Sci.  Philadelphia,  Ivi 
(1904)  p.  475;  Jour.  Roy.  Micr.  Soc.  1905,  p.  103)  a  j  in.  objective  made  by 
Chas.  A.  Spencer  in  1860  contained  a  fluorite  lens  in  one  of  the  combinations. 


i6 


MICROSCOPE  AND  ACCESSORIES 


\CH.1 


\  26.  Non- Adjustable  or  Unadjustable  Objectives. — Objectives  in  which 
the  lenses  or  lens  systems  are  permanently  fixed  in  their  mounting  so  that 
their  relative  position  always  remains  the  same.  Lower  power  objectives  and 
those  with  homogenous  immersion  are  mostly  non-adjustable.  For  beginners 
and  those  unskilled  in  manipulating  adjustable  objectives  (§  27),  non-adjusta- 
ble ones  are  more  satisfactory,  as  the  optician  has  put  the  lenses  in  such  a 
position  that  the  most  satisfactory  results  may  be  obtained  when  the  proper 
thickness  of  cover-glass  and  tube-length  are  employed  (See  table  of  tube- 
length  and  thickness  of  cover-glass  below  ($  34). 

I  27.  Adjustable  Objectives. — An  adjustable  objective  is  one  in  which 
the  distance  between  the  systems  of  lenses  (usually  the  front  and  the  back 
systems)  may  be  changed  by  the  observer  at  pleasure.  The  object  of  this 
adjustment  is  to  correct  or  compensate  for  the  displacement  of  the  rays  of 
light  produced  by  the  mounting  medium  and  the  cover-glass  after  the  rays 
have  left  the  object.  It  is  also  to  compensate  for  variations  in  ''  tube-length". 
See  \  32.  As  the  displacement  of  the  rays  by  the  cover-glass  is  the  most  con- 
stant and  important,  these  objectives  are  usually  designated  as  having  cover- 
glass  adjustment  or  correction.  (Fig.  28.  See  also  practical  work  with 
adjustable  objectives,  Ch.  II.) 

$  28  .  Parachromatic,  Pantachromatic  and  Semi-apochromatic  Objec- 
tives.— These  are  trade  names  for  objectives,  most  of  them  containing  one  or 
more  lenses  of  the  new  glass  ( f  25) .  They  are  said  to  approximate  much 
more  closely  to  the  apochromatics  than  to  the  ordinary  objectives. 

$  29.  Variable  Objective. — This  is  a  low  power  objective  of  36  to  26  mm. 
equivalent  focus,  depending  upon  the  position  of  the  combinations.  By  means 
of  a  screw  collar  the  combinations  may  be  separated,  diminishing  the  power,  or 
approximated  and  thereby  increasing  it. 

FIG.  29.  An  objective  in  section,  showing  the  differ- 
ent combinations  formed  of  concave  and  convex  lenses. 
Cut  loaned  by  Voigtldnder  &  So/in,  A.  G. 

\  30.  Projection  Objectives. — These  are  designed 
especially  for  projecting  an  image  on  a  screen  and  for 
photo-micrography.  They  are  characterized  by  having 
a  flat,  sharp  field  brilliantly  lighted.  (See  Ch.  IV,  IX.) 

§  31.  Illuminating  or  Vertical  Illuminating  Ob- 
jectives.— These  are  designed  for  the  study  of  opaque 
objects  with  good  reflecting  surfaces,  like  the  rulings  on 
metal  bars  and  broken  or  polished  and  etched  surfaces  of  metals  employed  in 
micro-metallography.  The  light  enters  the  side  of  the  tube  or  objective  and 
is  reflected  vertically  downward  through  the  objective  and  thereby  is  concen- 
trated upon  the  object.  The  object  reflects  part  of  the  light  back  into  the 
microscope  thus  enabling  one  to  see  a  clear  image.  For  a  figure  see  Ch.  VIII. 


CH.  /]  MICROSCOPE  AND  ACCESSORIES  17 

\  32.  Tube-length. — "In  the  construction  of  microscopic  objectives,  the 
corrections  must  be  made  for  the  formation  of  the  image  at  a  definite  distance, 
or  in  other  words  the  tube  of  the  microscope  on  which  the  objective  is  to  be 
used  must  have  a  definite  length.  Consequently  the  microscopist  must  know 
and  use  this  distance  or  '  microscopical  tube-length  '  to  obtain  the  best  results 
in  using  any  objective  in  practical  work."  Unfortunately  different  opticians 
have  selected  different  tube-lengths  and  also  different  points  between  which 
the  distance  is  measured,  so  that  one  must  know  what  is  meant  by  the  tube- 
length  of  each  optician  whose  objectives  are  used.  See  table,  \  34. 

\  33.  The  Thickness  of  Cover-glass  used  on  an  object  (See  Ch.  VII,  on 
mounting),  except  with  homogeneous  immersion  objectives,  has  a  marked 
effect  on  the  light  passing  from  the  object  (Fig.  64).  To  compensate  for  this 
the  position  of  the  systems  composing  the  objective  are  closer  together  than 
they  would  be  if  the  object  were  uncovered.  Consequently  in  non-adjustable 
objectives  some  standard  thickness  of  cover-glass  is  chosen  by  each  optician 
and  the  position  of  the  systems  arranged  accordingly.  With  such  an  objective 
the  image  of  an  uncovered  object  would  be  less  distinct  than  a  covered  one, 
and  the  same  result  would  follow  the  use  of  a  cover-glass  much  too  thick. 

\  34.  In  the  following  tables  tube-length  b-d  of  the  diagram  greatly  pre- 
ponderates, and  a  large  majority  of  unadjustable  objectives  are  corrected  for  a 
thickness  of  cover-glass  falling  between  fifteen  and  twenty  hundredths  of  a 
millimeter  (0.15-0.20  mm.). 


*  The  information  contained  in  the  tables  on  the  following  page  was  very 
kindly  furnished  by  the  opticians  named,  or  obtained  by  consulting  catalogs. 
In  most  of  the  later  catalogs  the  information  is  definite,  and  many  makers 
now  not  only  put  their  names  and  the  equivalent  focal  length  on  their  objec- 
tives, but  they  add  the  numerical  aperture  (\  36)  and  the  tube-length  for 
which  the  objective  is  corrected.  This  is  in  accordance  with  the  recommenda- 
tions of  the  author  in  the  original  paper  on  "tube-length,"  (Proc.  Amer.  Soc. 
Micr.,  Vol.  IX.,  p.  168,  also  by  Edward  Bausch,  Vol.  XII.,  p.  43).  If  the 
table  in  this  edition  is  compared  with  the  original  table  or  with  that  in  the 
previous  editions  of  this  book  some  differences  will  be  noted,  the  changes 
being  in  the  direction  of  uniformity  and  in  general  in  the  direction  recom- 
mended by  the  writer  and  Mr.  Edward  Bausch  and  the  committee  of  the  Amer- 
ican Microscopical  Society.  The  recommendations  of  the  committee  were 
published  in  the  Proceedings,  Vol.  XII.  p.  250. 


i8 


MICROSCOPE  AND  ACCESSORIES 


\CH.l 


e 


Length  in  Millimeters  and  Parts  included  in  the  "  Tube-Length  "  by 
Various   Opticians. 

Pts.  included 

in  "Tube-  "  Tube-Length  "  in 

Length."  Millimeters. 

See  Diagram. 

f  Chas.  Baker, -London,  England •___. 150  or  250  mm. 

'  The  Bausch  &  Lomb  Optical  Co., 

Rochester,  N.  Y 160  mm. 

R.  &  J.  Beck,  London,  England 160  or  220  mm. 

Bezu,  Hausser  &  Cie,  Paris,  France 180  mm. 

|  Klonne  und  Miiller,  Berlin,  Germany 160  or  250  mm. 

b-d<i|  Queen  &  Co.,  Incorporated,  Phila.,  Pa. 170  mm. 

|  Ross,  Ltd,  London,  England i6oor  254mm. 

I  W.  und  H.  Seibert,  Wetzlar,  Germany,  170  mm. 

|  Swift  &  Son,  London,  England 160  or  228  mm. 

|  Voigtlander  und  Sohn,  A.  G 160  mm. 

|  Watson  &  Sons,  London,  England 160  or  250  mm. 

j  R.  Winkel,  Goettingen,  Germany 192  mm. 

[Carl  Zeiss,  Jena,  Germany 160  or  250  mm. 

f  Ernst  Leitz,  Wetzlar,  Germany 170  mm. 

|  Nachet  et  Fils,  Paris,  France 160  mm. 

a-d  -\  Powell  &  Lealand,  London,  England 254  mm. 

|  C.  Reichert,  Vienna,  Austria 160-180  mm. 

[Spencer  Lens  Company,  Buffalo,  N.  Y.  160  mm. 

f  E.  Hartnack,  Potsdam,  Germany 160  mm. 

]  Dollond  &  Co.,  London,  England 165,  240  nun. 

IlVIg    c-f  -j  Verick  (Stiassnie)  Paris,  France 160-200  mm. 

|  P.  Wachter,  Berlin-Friedenau,  Germanyi6o  mm. 
jG   ,Q  [J.  Zentmayer,  Philadelphia,  Pa 160  or  235  mm. 

Thickness  of  Cover-Glass  for  Which  Non-Adjiistable  Objectives  are  Corrected 

by  Various  Opticians. 

f  The  Bausch  &  Lomb  Optical  Co  ,  Rochester,  N.  Y. 
|  Klonne  und  Miiller,  Berlin,  Germany. 

0.18  mm.  \  Queen  &  Co.,  Incorporated,  Philadelphia,  Pa. 

I  The  Spencer  Lens  Co.,  Buffalo,  N.  Y. 
[Voigtlander  und  Sohn,  A.  G.  Brunswick,  Germany. 

{Ernst  Leitz,  Wetzlar,  Germany.  . 

P.  Wachter,  Berlin-Friedenau,  Germany. 
R.  Winkel,  Goettingen,  Germany. 
(Chas.  Baker,  London,  England. 
R.  &  J.  Beck,  Ltd.,  London,  England. 
W.  und  H.  Seibert,  Wetzlar,  Germany. 
f  E.  Hartnack,  Potsdam,  Germany. 
0.15-0.  IS     im.       \  c.  Reichert,  Vienna,  Austria. 
( Ros«,  Ltd.,  London,  England. 
0.15-0. 20  mm.       \  Verick  (Stiassnie),  Paris,  France. 

(  Carl  Zeiss,  Jena,  Germany. 
0.12-0.17  mm.          J.  Zentmayer,  Philadelphia,  Pa. 

f  Dollond  &  Co.,  London,  England, 
o.  i  o-o. 15  mm.       J  Nachet  et  Fils,  Paris,  France. 
0.10-0.12  mm.          Bezu  Hausser  &  Cie,  Paris,  France. 

/  Powell  &  Lealand,  London,  England. 
°'10  \  Swift  &  Son,  London,  England. 

0.20  mm.  Watson  &  Sons,  London,  England. 


CH.  7] 


MICROSCOPE  AND   ACCESSORIES 


2  35.  Aperture  of  Objectives. — The  angular  aperture  or  angle  of  aperture 
of  an  objective  is  the  "  angle  contained,  in  each  case,  between  the  most  diverg 
ing  of  the  rays  issuing  from  the  axial  point  of  an  object  [z.  e. ,  a  point  in  the 
object  situated  on  the  optic  axis  of  the  microscope] ,  that  can  enter  the  objec- 
tive and  take  part  in  the  formation  of  an  image."  (Carpenter. ) 

In  general  the  angle  increases  with  the  size  of  the  lenses  forming  the 
objective  and  the  shortness  of  the  equivalent  focal  distance  (§  18).  If  all 


FIG.  31.  The  tube  of  a  micro- 
scope with  ocular  micrometer  and 
nose  piece  in  position  to  show  that  in 
measuring  tube-length  one  must 
measure  from  the  eye  lens  to  the  place 
where  the  objective  is  attached. 
(Zeiss'  Catalog.} 


objectives  were  dry  or  all  water  or  all  homogeneous  immersion  a  comparison 
of  the  angular  aperture  would  give'one  a  good  idea  of  the  relative  number  of 
image  forming  rays  transmitted  by  different  objectives  ;  but  as  some  are  dry, 
others  water  and  still  others  homogeneous  immersion,  one  can  see  at  a  glance 
that,  other  things  being  equal,  the  dry  objective  (Fig  33)  receives  lesg  light 

FIG.  32.  Diagram  illustrating  the  angular  aper- 
ture of  a  microscopic  objective.  Only  the  front  lens  of 
the  objective  is  shown. 

A.ris.     Theprincipal  optic  a.vis  of  the  objective. 

B  A,  B  C,  the  most  divergent  rays  that  can  enter 
the  objective,  they  mark  the  angular  aperture.  A  B  D 
or  C  B  D  half  the  angular  aperture.  This  is  designated 
by  u  in  making  Numerical  Aperture  computations. 
Seethe  table,  (\  39). 

than  the  water  immersion,  and  the  water  immersion  (Fig.  34)  less  than  the 
homogeneous  immersion  (Fig.  35).  In  order  to  render  comparison  accurate 
between  different  kinds  of  objectives,  Professor  Abbe  takes  into  consideration 


2o  ^rlCROSCOPE  AND  ACCESSORIES  [  CH.  i 

the  rays  actually  passing  from  the  back  combination  of  the  objectives  to  form 
the  real  image  ;  he  thus  takes  into  account  the  medium  in  front  of  the  objec- 
tive as  well  as  the  angular  aperture.  The  term  "Numerical  Aperture" 
(2V.  A.}  was  introduced  by  Abbe  to  indicate  the  capacity  of  an  optical  instru- 
ment "  for  receiving  rays  from  the  object  and  transmitting  them  to  the  image". 

\  36.  Numerical  Aperture  (abbreviated  N.  A.),  as  now  employed  for 
microscope  objectives,  is  the  ratio  of  the  semi-diameter  of  the  emergent  pencil 
to  the  focal  length  of  the  lens.  Or  as  the  factors  are  more  readily  obtainable 
it  is  simpler  to  utilize  the  relationship  shown  in  the  La  Grange-Helmholtz- 
Abbe  formula,  and  indicate  the  aperture  by  the  expression:  N.  A.=«  sin  u. 
In  this  formula  n  is  the  index  of  refraction  of  the  medium  in  front  of  the 
objective  (air,  water  or  homogeneous  liquid),  sin  u  is  thesine  of  half  theangle 
of  aperture  (Fig.  32,  D  B  A).  For  the  mathematical  discussion  showing  that 
the  expressions 

semi-diameter  of  emergent  pencil 

— ? i-: —       — 7—. -, —  — =n  sin  u,  the  student  is  referred  to  the  four- 

focal  length  of  the  lens 

nal  of  the  Royal  Microscopical  Society,  1881,  pp.  392-395,  1898,  p.  363. 

\  37.  Comparison  of  Dry  and  Immersion  Objectives. — For  example,  take 
three  objectives  each  of  3  mm.  equivalent  focus,  one  being  a  dry,  one  a  water 
immersion,  and  one  a  homogeneous  immersion.  Suppose  that  the  dry  objec- 
tive has  an  angular  aperture  of  106°,  the  water  immersion  of  94°  and  the  homo- 
geneous immersion  of  90°.  Simply  compared  as  to  their  angular  aperture, 
without  regard  to  the  medium  in  front  of  the  objective,  it  would  look  as  if  the 
dry  objective  would  actually  take  in  and  transmit  a  wider  pencil  of  light  than 
either  of  the  others.  However,  if  the  medium  in  front  of  the  objective  is 
considered,  that  is  to  say,  if  the  numerical  instead  of  the  angular  apertures  are 
compared,  the  results  would  be  as  follows :  Numerical  Aperture  of  a  dry  ob- 
jective of  106°,  N.  A.=»  sin  u.  In  the  case  of  dry  objectives  the  medium  in 
front  of  the  objective  being  air,  the  index  of  refraction  is  unity,  whence  »=1. 
Half  the  angular  aperature  is  isp°=53°.  By  consulting  a  table  of  natural 
sines  it  will  be  found  that  the  sine  of  53°  is  0.799,  whence  N.  A.=«  or  1  x  sin 
u  or  0.799=0.799.* 


*\  38.  Interpolation. — In  practice,  as  in  solving  problems  similar  to  those 
on  the  following  pages  and  those  in  refraction  if  one  cannot  find  a  sine  exactly 
corresponding  to  a  given  angle  ;  or  if  one  has  an  angle  which  does  not  corres- 
pond to  any  sine  or  angle  given  in  the  table,  the  sine  or  angle  may  be  closely 
approximated  by  the  method  of  interpolation,  as  follows  :  Find  the  sine  in  the 
table  nearest  the  sine  whose  angle  is  to  be  determined.  Get  the  difference  of 
the  sines  of  the  angles  greater  and  less  than  the  sine  whose  angle  is  to  be  de- 
termined. That  will  give  the  increase  of  sine  for  that  region  of  the  arc  for  15 
minutes.  Divide  this  increase  by  15  and  it  will  give  with  approximate  accur- 
acy the  increase  for  i  minute.  Now  get  the  difference  between  the  sine  whose 
angle  is  to  be  determined  and  the  sine  just  below  it  in  value.  Divide  this 
difference  by  the  amount  found  necessary  for  an  increase  in  angle  of  i  minute 
and  the  quotient  will  give  the  number  of  minutes  the  sine  is  greater  than  the 


CH.I} 


MICROSCOPE  AND  ACCESSORIES 


21 


FIGS.  33,  34,  35  are  somewhat  modified  from  Ellenberger,  and  are 
introduced  to  illustrate  the  relative  amount  of  utilized  light,  with  dry,  water 
immersion  and  homogeneous  immersion  objectives  of  the  same  equivalent 
focus.  The  point  from  <i>/iich  the  rays  emanate  is  in  air  in  each  case. 
If  Canada  balsam  r<r;r  beneath  the  cover-glass  in  place  of  the  air 

their  would  be  practically  no  refraction 
of  the  ravs  on  entering  the  cover-glass 


FIG.  33.  Showing  the  course  of  the 
rays  passing  through  a  cover-glass  from 
an  a. rial  point  of  the  object,  and  the  num- 
ber that  finally  enter  the  front  of  a  dry 
objective. 


FIG.  34.  Rays  from  the  axial  point 
of  the  object  traversing  a  cover  of  the 
same  thickness  as  in  Fig.  jj,  and  entering 
the  front  lens  of  a  water  immersion 
objective. 


FIG.  35.  Rays  from  an  a. rial  point 
of  the  object  traversing  a  cover-glass  and 
entering  the  front  of  a  homogeneous  im- 
mersion objective. 


next  lower  sine  whose  angle  is  known.  Add  this  number  of  minutes  to  the 
angle  of  the  next  lower  sine  and  the  sum  will  represent  the  desired  angle. 
Or  if  the  sine  whose  angle  is  to  be  found  is  nearer  in  size  to  the  sine 
just  greater,  proceed  exactly  as  before,  getting  the  difference  in  the  sines, 
but  subtract  the  number  of  minutes  of  difference  and  the  result  will  give  the 
angle  sought.  For  example  take  the  case  in  Section  108  where  the  sine  of  the 
angle  of  28°  54'  is  given  as  0.48327.  If  one  consults  the  table  the  nearest  sines 
found  are  0.48099,  the  sine  of  28°  45',  and  0.4848 r,  the  sine  of  29°.  Evidently 
then  the  angle  sought  must  lie  between  28°  45',  and  29°.  If  the  difference 
between  o  48481  and  0.48099  is  obtained,  0.48481 — 0.48099—0.00382,  and  if  this 
increase  for  15'  be  divided  by  15  it  will  give  the  increase  for  i  minute ; 
o.oo382-=-i5=o.ooo254.  Now  the  difference  between  the  sine  whose  angle  is  to 
be  found  and  the  next  lower  sine  is  0.48327 — 0.48099=0  00228.  If  this  differ- 
ence be  divided  by  the  amount  found  necessary  for  i  minute  it  will  give  the 
total  minutes  above  28°  45',  0.00228^-0.000254—9.  That  is,  the  angle  sought  is 
9  minutes  greater  than  28°  45/=2S°  54'. 


22  MICROSCOPE  AND  ACCESSORIES  [  CH.  I 

With  the  water  immersion  objective  the  medium  in  front  is  water,  and  its 
index  of  refraction  is  1.33,  whence  «=i.33.  Half  the  angular  aperture  is 
y°=47°,  and  by  the  table  the  sine  of  47°  is  found  to  be  0.731,  i.  c.,  sin  u= 
0.731,  whence  N.  A.=»  or  i.33Xsin  u  or  0.731=0.972. 

With  the  oil  immersion  in  the  same  way  N.  A.=w  sin  u  ;  n  or  the  index 
of  refraction  of  the  homegeneous  fluid  in  front  of  the  objective  is  1.52,  and  the 
semi-angle  of  aperture  is  -920-0=450.  The  sine  of  45°  is  0.707,  whence  X.  A.=# 
or  i.52Xsin  u  or  0.707=1.074. 

By  comparing  these  numerical  apertures:  Dry  0.799,  water  0.972,  homo- 
geneous immersion  1.074,  the  same  idea  of  the  real  light  efficiency  and  image 
power  of  the  different  objectives  is  obtained,  as  in  the  graphic  representations 
shown  in  Figs.  33-35. 

If  one  knows  the  numerical  aperture  (N.  A.)  of  an  objective  the  angular 
aperture  is  readily  determined  from  the  formula  ;  and  one  can  determine  the 
equivalent  angles  of  objectives  used  in  different  media  (i.  e.,  dry  or  immer- 
sion). For  example,  suppose  each  of  three  objectives  has  a  numerical  aper- 
ture (N.  A.)  of  0.80,  what  is  the  angular  aperture  of  each?  Using  the  formula 
of  N.  A.=«  sin  u,  one  has  N.  A=o.So  for  all  the  objectives. 
For  the  dry  objective  »=i  (Refractive  index  of  air). 

For  the  water  immersion  objective  w=i.33  ( Refractive  index  of  water). 

For  the  homogeneous  immersion  objective  #=1.52  (Refractive  index  of 
homogeneous  liquid).  And  2  u  is  to  be  found  in  each  case. 

For  the  dry  objective,  substituting  the  known  values  the  formula  becomes 
0.80=1  sin  u,  or  sin  #=o.8o.  By  inspecting  the  table  of  natural  sines  (3d  page 
of  cover)  it  will  be  found  that  0.80  is  the  sine  of  53  degrees  and  8  minutes. 
As  this  is  half  the  angle  the  entire  angular  aperture  of  the  dry  objective  must 
be  53°  8/X2=io6°  16'. 

For  the  water  immersion  objective,  substituting  the  known  values  in  the 

0.80  , 

formula  as  before  :  0.80=1.33  sin  u,  or  sin  «=        -=0.6015. 

1-33 

Consulting  the  table  of  sines  as  before,  it  will  be  found  that  0.6015  is  the  sine 
of  36°  59'  whence  the  angular  aperture  (water  angle)  is  36°  59/X2=73°  58'. 

For  the  homogeneous  immersion  objective,  substituting  the  known  values, 

0.80 
the  formula  becomes  :  0.80=1.52  sin  u  whence  sin  #=-       =0.5263.      And   by 

1-52 

consulting  the  table  of  sines  it  will  be  found  that  this  is  the  sine  of  31°  45%' 
whence  2  u  or  the  entire  angle  (balsam  or  oil  angle)  is  63°  31'. 

That  is,  three  objectives  of  equal  resolving  powers,  each  with  a  numerical 
aperture  of  0.80  would  have  an  angular  aperture  of  106°  i6x  in  air,  73°  58'  in 
water  and  63°  31'  in  homogeneous  immersion  liquid. 

For  the  apparatus  and  method  of  determining  aperture,  see  Ch.  X. 

\  39.  Table  of  a  group  of  Objectives  with  the  Numerical  Aperture  (N.  A.) 
and  the  method  of  obtaining  it-.  Half  the  angular  aperture  is  designated  by  u 
and  the  index  of  refraction  of  the  medium  in  front  of  the  objective  by  n.  For 


CH.  /] 


MICROSCOPE  AND  ACCESSORIES 


dry  objectives  t/iis  is  air  and  n=/,for  water  immersions  n -.--/.  y(  and  for 
homogeneous  immersions  11=1.52.  (For  a  table  of  natural  sines,  see  third 
page  of  coi't •;-. ) 


u 

I-  p  .-^ 

OBJECTIVE     -2  -  « 

NATURAL  SINE 
of  half  the  angular 
aperture 

(sin  u.  } 

Index    of 
Refraction 
of  the   medi- 
um in  front 
of  the  objec- 
tive (  »  ) 

NUMERICAL  APERTURE 
(N.  A.)=  n  sin  u 

25D?ym'              20°          Sin—  -0.1736 

n     i 

N.A.=:    1X0.1736=0.173 

25  mm. 
Dry. 

40° 

40 
Sin  =  0.3420 

n=.\ 

N.A.=    1X0.3420=0.342 

\2l/2  mm. 
Dry. 

42° 

42 

Sin  =0.3584 

2 

—. 

N.A.=    1X0.3583=0.358 

12^  mm. 
Dry. 

100° 

IOO 

Sin  =0.7660 

2 

n     i 

N.A.=    1X0.7660=0.766 

6  mm. 
Dry. 

75° 

75 
Sin  =0.6087 

2 

*=, 

N.A.=    1X0.6087=0.609 

6  mm. 
Dry. 

136° 

136 

Sin  =0.9272 

2 

„=, 

N.A.=    1X0.9272=0.927 

3  mm. 
Dry. 

115° 

Sin  —    =0.8434 

n=i 

N.A.=    1X0.8434=0.843 

3  mm. 
Dry. 

163° 

163 
Sin  =0.9890 

2 

n=i 

N.A.=    1X0.9890=0989 

2  mm. 
Water 
Immersion 

<)6°  12' 

96°  12' 

2 

n  —  J>33 

2  mm. 
Homogeneous 
Immersion 

i  io°38' 

no°38/ 

2 

2  mm. 
Homogeneous 
Immersion 

,34- 

i34°io' 

N  A  —  I  ^2X0  0210^^1  40 

2 

\  40.  Significance  of  Aperture. — As  to  the  real  significance  of  aperture  in 
microscopic  objectives,  it  is  now  an  accepted  doctrine  that — the  corrections  in 
spherical  and  chromatic  aberration  being  the  same — (i)  Objectives  vary 


24  MICROSCOPE  AND  ACCESSORIES  [  CH.  1 

directly  as  their  numerical  aperture  in  their  ability  to  define  or  make  clearly 
visible  minute  details  (resolving  power).  For  example  an  objective  of  4  mm. 
equivalent  focus  and  a  numerical  aperture  of  0.50  would  define  or  resolve 
only  half  as  many  lines  to  the  millimeter  or  inch  as  a  similar  objective  of  i.oo 
N.A.  So  also  an  objective  of  2  mm.  focus  and  1.40  N.A.  would  resolve 
only  twice  as  many  lines  to  the  millimeter  as  a  4  mm.  objective  of  0.70  N.A. 
Thus  it  is  seen  that  defining  power  is  not  a  result  of  magnification  but  of 
aperture,  otherwise  the  2  mm.  objective  would  resolve  far  more  than  twice  as 
many  lines  as  the  4  mm.  objective. 

Taking  the  results  of  the  researches  of  Abbe  as  a  guide  to  visibility  with 
the  microscope,  one  has  the  general  formula  2/1 X N.A.  That  is  twice  the  num- 
ber of  wave  lengths  of  the  light  used  multiplied  by  the  numerical  aperture  of 
the  objective.  From  this  general  statement  it  will  be  seen  that  the  shorter  the 
wave  lengths  of  the  light,  the  more  there  will  be  in  an  inch  or  centimeter  and 
therefore  the  greater  the  number  of  lines  visible  in  a  given  space.  That  is  the 
kind  of  light  used  is  one  element  and  the  objective  the  other  in  determining 
the  number  of  lines  visible  under  the  microscope. 

Following  Mr.  E.  M.  Nelson  (Jour.  Roy.  Micr.  Soc. ,  1893,  p.  15,  and  1906, 
p.  521)  it  is  believed  that  not  more  than  %  of  the  numerical  aperture  of  an 
objective  is  really  available  for  microscopic  study,  with  a  central,  solid  cone  of 
light.  To  determine  the  number  of  lines  visible  in  a  given  space  with  a  given 
light  the  formula  would  become  2/lX^N.A.=3/2AN.A.  To  determine  the 
working-resolving  power  of  any  objective  it  is  only  necessary  to  know  the 
number  of  light  waves  in  a  given  space,  say  an  inch  or  a  centimeter  and  to 
multiply  this  number  by  3/aN.A.  For  example  suppose  one  uses  ordinary 
daylight  and  assumes  the  average  wave  length  is  1/46666  in.,  then  there  must 
be  46,666  per  inch  and  46,666x3/2=70,000  approximately.  If  the  N.A.  is  i, 
then  the  objective  will  resolve  or  make  visible  70,000  lines  to  the  inch,  or 
approximately  28,000  to  the  centimeter.  If  blue  light  were  used  the  number 
would  be  32,000  per  centimeter,  or  80,000  per  inch.  It  will  be  seen  that  the 
number  of  lines  here  given  is  smaller  than  that  in  the  table  of  Carpenter- 
Dalliuger,  because  in  the  latter  the  full  aperture  is  supposed  to  be  employed 
and  the  light  is  of  the  greatest  available  obliquity,  while  here  only  34  of  the 
aperture  is  assumed  to  be  available. 

(2)  The  illuminating  power  of  an  objective  of  a  given  focus  is  found  to 
vary  directly  as  the  square  of  the  numerical  aperture   (N.A.)2.     Thus  if  two 
4  mm.  objectives  of  N.A.  0.20  and  N.A.  0.40  were  compared  as  to  their  illumi- 
nating power  it  would  be   found   from   the   above  that   they  would  vary  as 
o.2o2:o.4o2=o.o4oo:o.i6oo  or  i  -.4.     That  is  the  objective  of  0.20  N.A.   would 
have  but  %  the  illuminating  power  of  the  one  of  0.40  N.A. 

(3)  The  penetrating  power,  that  is  the  power  to  see  more  than  one  plane, 

i 
is  found  to  vary  as  the  reciprocal  of  the  numerical  aperture  —      —  so  that  in 

N.A. 

an  objective  of  a  given  focus  the  greater  the  aperture  the  less  the  penetrating 
power. 


i  II.  /]  MICROSCOPE  AND  ACCESSORIES  25 

Of  course  when  equivalent  focus  and  numerical  aperture  both  differ  the 
problem  becomes  more  complex. 

While  all  niicroscopists  are  agreed  that  the  fineness  of  detail  which  can 
be  seen  depends  directly  on  the  numerical  aperture  of  the  objective  used,  the 
general  theory  of  microscopic  vision  has  two  interpretations  : 

(A)  That  it  is  as  with  the  unaided  eye,  the  telescope   and   the   photo- 
graphic  camera.       This   is  the   original  view  and  the  one  which  many  are 
favoring  at  the  present  day  (see  Mercer,  Proceedings  of  the  Amer.  Micr.  Soc. 
1896,  pp.  321-396  ;  Wright,  Gordon  and  Beck). 

(B)  The  other  view  originated  with  Professor  Abbe,  and  in  the  words  of 
Carpenter-Dallinger,  pp.  62,  43:    "What  this  is  becomes  explicable  by  the 
researches  of  Abbe.     It  is  demonstrated  that  microscopic  vision  is  sui  generis. 
There  is  and  can  be,  no  comparison  between  microscopic   and   macroscopic 
vision.     The  images  of  minute  objects  are  not  delineated  microscopically  by 
means  of  the  ordinary  laws  of  refraction  ;  they  are  not  dioptrical  results,  but 
depend  entirely  on  the  laws  of  diffraction.     These  come  within  the  scope  of 
and  demonstrate  the  undulatory  theory  of  light,  and  involve  a  characteristic 
change  which  material  particles  or  fine  structural  details,  in  proportion  to 
their  minuteness,  effect  in   transmitted  rays  of  light.     The  change  consists 
generally  in  the  breaking  up  of  an  incident  ray  into  a  group  of  rays  with 
large  angular  dispersion  within  the  range  of  which  periodic  alternations  of 
dark  and  light  occur." 

For  a  consideration  of  the  aperture  question,  its  history  and  significance, 
see  J.  D.  Cox,  Proc.  Amer.  Micr.  Soc.,  1884,  pp.  5-39;  Jour.  Roy.  Micr.  Soc., 
i88r,  pp.  303,  348,  365,  388;  1882,  pp.  300,  4bo;  1883,  p.  790;  1884,  p.  20; 
1896,  p.  681  ;  1897,  p.  71  ;  1898,  pp.  354,  362,  592;  Mercer,  Proceedings  Amer. 
Micr.  Soc.,  1896,  pp.  321-396;  Lewis  Wright,  Philos.  Mag. ,  June,  1898,  pp. 
480-503  ;  Carpenter-Dallinger,  Chapter  II  ;  Nelson,  Jour.  Quekett  Micr.  Club, 
VI,  pp.  14-38  ;  Jour.  Roy.  Micr.  Soc.,  1906,  pp.  521-531  ;  A.  E.  Wright's  Prin- 
ciples of  Microscopy  ;  Conrad  Beck,  Theory  of  the  Microscope.  Gordon, 
Jour.  Roy.  Micr.  Soc.,  1902. 

THE   OCULAR 

$  41.  A  Microscopic  Ocular  or  Eye-Piece  consists  of  one  or  more  con- 
verging lenses  or  lens  systems,  the  combined  action  of  which  is,  like  that  of  a 
simple  microscope,  to  magnify  the  real  image  formed  by  the  objective. 

Depending  upon  the  relation  and  action  of  the  different  lenses  form- 
ing oculars,  they  are  divided  into  two  great  groups,  negative  and  positive. 

$  42.  Negative  Oculars  are  those  in  which  the  real,  inverted  image  is 
formed  within  the  ocular,  the  lower  or  field-lens  serving  to  collect  the  image- 
forming  rays  somewhat,  so  that  the  real  image  is  smaller  than  as  if  the  field- 
lens  were  absent  (Fig.  26).  As  the  field-lens  of  the  ocular  aids  in  the  forma- 
tion of  the  real  image  it  is  considered  by  some  to  form  a  part  of  the  objective 
rather  than  of  the  ocular.  The  upper  or  eye-lens  of  the  ocular  magnifies  the 
real  image. 


26 


MICROSCOPE  AND  ACCESSORIES 


\_Cff.  I 


I  43.  Positive  Oculars  are  those  in  which  the  real,  inverted  image  of  the 
object  is  formed  outside  the  ocular,  and  the  entire  system  of  ocular  lenses 
magnifies  the  real  image  like  a  simple  microscope  (Fig.  16). 

Positive  and  negative  oculars  may  be  readily  distinguished,  as  the  dia- 
phragm is  below  the  ocular  lenses  with  the  positive  ocular  and  between  the 
lenses  in  the  negative  ocular  (Figs.  36-37). 


FIG.  36.  Sectional  view  of  a  Huygenian  ocular  to 
show  the  formation  of  the  Eye-Point. 

Axis.  Optic  axis  of  the  ocular.  D.  Diaphragm 
of  the  ocular.  E.  L.  Eye-Lens.  F.  L.  Field-Lens. 

E.  P.  Eye-Point.  As  seen  in  section,  it  appears 
something  like  an  hour-glass.  When  seen  as  looking 
into  the  ocular,  i.  e.,  in  transection,  it  appears  as  a  cir- 
cle of  light.  It  is  at  the  point  where  the  most  rays  cross. 


TABLE    OF   OCULARS 

§  44.  In  works  and  catalogs  concerning  the  microscope  and  microscopic 
apparatus,  and  in  articles  upon  the  microscope  in  periodicals,  various  forms  of 
oculars  or  eye-pieces  are  so  frequently  mentioned,  without  explanation  or 
definition,  that  it  seems  worth  while  to  give  a  list,  with  the  French  and  Ger- 
man equivalents,  and  a  brief  statement  of  their  character. 

Achromatic  Ocular;  Fr.  Oculaireachromatique;  Ger.  achromatisches  Oku- 
lar.  Oculars  in  which  chromatic  aberation  is  wholly  or  nearly  eliminated. — 
Aplanatic  Ocular;  Fr.  Oculaire  aplanatique;  Ger.  aplanatisches  Okular  (see 
§  24) . — Binocular,  stereoscopic  Ocular;  Fr.  Oculaire  binoculaire  stereoscopique; 
Ger.  stereoskopisches  Doppel-Okular.  An  ocular  consisting  of  two  oculars 
about  as  far  apart  as  the  two  eyes.  These  are  connected  with  a  single  tube 
which  fits  a  monocular  microscope.  By  an  arrangement  of  prisms  the  image 
forming  rays  are  divided,  half  being  sent  to  each  eye.  The  most  satisfactory 
form  was  worked  out  by  Tolles  and  is  constructed  on  true  stereotomic  princi- 
ples, both  fields  being  equally  illuminated.  His  ocular  is  also  erecting. — 
Campani" 's  Ocular  (see  Huygenian  Ocular). — Compound  Ocular;  Fr.  Oculaire 
compose;  Ger.  zusammengesetztes  Okular.  An  ocular  of  two  or  more  lenses, 
e.  g.,  the  Huygenian  (see  Fig.  36). — Continental  Ocular.  An  ocular  mounted 
in  a  tube  of  uniform  diameter  as  in  Fig.  37. — Deep  Ocular,  see  high  ocular. — 
Erecting  Ocular;  Fr.  Oculaire  redresseur;  Ger.  bildumkehrendes  Okular.  An 
ocular  with  which  an  erecting  prism  is  connected  so  that  the  image  is  erect  as 
with  the  simple  microscope.  Such  oculars  are  most  common  on  dissecting 
microscopes. — Filar  micrometer  Ocular;  Screw  m.  o.,  Cobweb  m.  o.,  Ger. 
Okular-Schraubenmikrometer.  A  modification  of  Ramsden's  Telescopic  Cob- 
web micrometer  ocular. — Goniometer  Ocular;  Fr.  Oculaire  a  goniometre;  Ger. 


\_CH.I  .MICROSCOPE  AND  ACCESSORIES  27 

Goniometer-Okular.  An  ocular  with  goniometer  for  measuring  the  angles  of 
minute  crystals. — High  Ocular,  sometimes  called  a  deep  ocular.  One  that 
magnifies  the  real  image  considerably,  i.  c. ,  10  to  20  fold. — Huygenian  Ocular, 
Huygens'  O.,  Campani's  O.,  Airy's  O.;  Fr.  Oculaire  d'Huygens,  o.  de  Cam- 
pani;  Ger.  Huygens'sches  Okular,  Campanisches  Okular,  see  \  45. — Index 
Ocular;  Ger,  Spitzen-O.  An  ocular  with  a  minute  pointer  or  two  pointers  at 
the  level  of  the  real  image.  The  points  are  movable  and  serve  for  indicators 
and  also,  although  not  satisfactorily,  for  micrometry. — A'cllner's  Ocular,  see 
orthoscopic  ocular — Low  ocular,  also  called  shallow  ocular.  An  ocular 
which  magnifies  the  real  image  only  moderately,  i.  e.,  2  to  8  fold. — 
Micrometer  or  micrometric  Ocular;  Fr.  Oculaire  micrometrique  ou  a 
micrometre;  Ger.  Mikrometer-Okular,  Mess  Okular  Beneches  O.,  Jack- 
son m.  o.,  see  $48. — Microscopic  Ocular;  Fr.  Oculaire  microscopique ; 
Ger.  mikroskopisches  Okular.  An  ocular  for  the  microscope  instead 
of  one  for  a  telescope. — Negative  Ocular,  see  \  42. — Nelson's  screw- 
micrometer  ocular.  A  modification  of  the  Ramsden's  screw  or  cob-web 
micrometer  in  which  positive  compensating  oculars  may  be  used. — Orthoscopic 
Oculars;  also  called  Kellner's  Ocular;  Fr.  Oculaire  orthoscopique;  Ger.  Kel- 
ner'sches  oder  orthoskopisches  Okular.  An  ocular  with  an  eye-lens  like  one 
of  the  combinations  of  an  objective  (Figs.  27,  29)  and  a  double  convex  field- 
lens.  The  field-lens  is  in  the  focus  of  the  eye-lens  and  there  is  no  diaphragm 
present.  The  field  is  large  and  flat. — Par-focal  Oculars,  a  series  of  oculars  so 
arranged  that  the  microscope  remains  in  focus  when  the  oculars  are  inter- 
changed (Pennock,  Micr.  Bulletin,  vol.  iii,  p.  9.  31,  1886). — Pe riscopic  Ocular; 
Fr.  Oculaire  periscopique  ;  Ger.  periskopisches  Okular.  A  positive  ocular 
devised  by  Gundlach.  It  consists  of  a  double  convex  field-lens  and  a  triplet 
eye-lens.  It  gives  a  large,  flat  field. — Positive  Ocular,  see  $  43. — Projection 
Ocular;  Fr.  Oculaire  de  projection;  Ger.  Projections-Okular,  see  §47. — 
Ramsden's  Ocular  ;  Fr.  Oculaire  de  Ramsden  ;  Ger.  Ramsden'sches  Okular. 
A  positive  ocular  devised  by  Ramsden.  It  consists  of  two  plano-convex  lenses 
placed  close  together  with  the  convex  surfaces  facing  each  other.  Only  the 
central  part  of  the  field  is  clear.  Searching  Ocular ;  Fr.  Oculaire  d'orienta- 
tion  ;  Ger.  Sucher-Okular,  see  \  46.  Shalloiv  Ocular,  see  low  ocular. — Solid 
Ocular,  holosteric  O. ;  Fr.  Oculaire  holostere ;  Ger.  holosterisches  Okular, 
Vollglass-Okular.  A  negative  eye-piece  devised  by  Tolles.  It  consists  of  a 
solid  piece  of  glass  with  a  moderate  curvature  at  one  end  for  a  field-lens,  and 
the  other  end  with  a  much  greater  curvature  for  an  eye- lens.  For  a  dia- 
phram,  a  groove  is  cut  at  a  proper  level  and  filled  with  black  pigment.  It  is 
especially  excellent  where  a  high  ocular  is  desired. — Spectral  or  spectroscopic 
Ocular ;  Fr.  Oculaire  spectroscopique  ;  Ger.  Spectral-Okular,  see  Microspec- 
troscope,  Ch.  VI. — Stauroscopic  Ocular  ;  Fr.  Oculaire  Stauroscopique ;  Ger. 
Stauroskop-Okular.  An  ocular  with  a  Bertrand's  quartz  plate  for  mineralog- 
cal  purposes — Working  Ocular;  Fr.  Oculaire  de  travail;  Ger.  Arbeits- 
Okular,  see  $  46. 

\  45.  Huygenian  Ocular. — A  negative  ocular  designed  by  Huygens  for 
the  telescope,  but  adapted  also  to  the  microscope.  It  is  the  one  now  most 
commonly  employed.  It  consists  of  a  field-lens  or  collective  (Fig.  36.  ),  aid- 


28 


MICROSCOPE  AND  ACCESSORIES 


CH.  /] 


ing  the  objective  in  forming  the  real  image,  and  an  eye-lens  which  magnifies 
the  real  image.  While  the  field-lens  aids  the  objective  in  the  formation  of 
the  real,  inverted  image,  and  increases  the  field  of  view,  it  also  combines  with 
the  eye-lens  in  rendering  the  image  achromatic. 


Oculir  lo  2 


FIG.  37.  Compensating  Oculars  of  Zeiss,  with  section  removed  to  show  the 
construction.  The  line  A- A  is  at  the  level  of  the  upper  end  of  the  tube  of  the 
microscope  while  B-B  represents  the  lower  focal  points.  It  will  be  seen  that 
the  mounting  is  so  arranged  that  the  lower  focal  points  in  all  are  in  the  same 
plane  and  therefore  the  microscope  remains  in  focus  upon  changing  oculars. 
( The  oculars  are  par-focal.)  The  lower  oculars  2,  4  and  6  are  negative,  and 
the  higher  ones,  8,  12,  18,  are  positive.  The  numbers  .?,  4,  6,  8,  12,  /£,  indicate 
the  magnification  of  the  ocular.  From  Zeiss'  Catalog. ) 

\  46.  Compensating  Oculars.— These  are  oculars  specially  constructed 
for  use  with  the  apochromatic  objectives.  They  compensate  for  aberrations 
outside  the  axis  which  could  not  be  so  readily  eliminated  in  the  objective  it- 
self. An  ocular  of  this  kind,  mangifying  but  twice,  is  made  for  use  with  high 
powers,  for  the  sake  of  the  large  field  in  finding  objects;  it  is  called  a  search- 
ing ocular;  those  ordinarily  used  for  observation  are  in  contradistinction 
called  working  octilars.  Part  of  the  compensating  oculars  are  positive  and 
part  negative.  (Fig.  37.) 

§  47.  Projection  Oculars. — These  are  oculars  especially  designed  for  pro- 
jecting a  microscopic  image  on  the  screen  for  class  demonstrations,  or  for 
photographing  with  the  microscope.  While  they  are  specially  adapted  for 
use  with  apochromatic  objectives,  they  may  also  be  used  with  ordinary  ach- 
romatic objectives  of  large  numerical  aperture.  The  projection  oculars 
(Fig.  38)  consist  of  a  collective  lens  or  field  lens  and  of  a  carefully  corrected 
system  for  the  eye  lens  The  eye  lens  is  movable  so  that  a  sharp  image  of 
the  diaphragm  between  the  field  and  eye  lens  may  be  projected  upon  the 
screen  at  different  screen  distances. 

\  48.     Micrometer  Ocular. — This  is  an  ocular  connected  with  an  ocular 


[CH.  I 


MICROSCOPE  AND  ACCESSORIES 


29 


micrometer.  The  micrometer  may  be  removable,  or  it  nay  be  permanently  in 
connection  with  the  ocular,  and  arranged  with  spring  and  screw,  by  which  it 
may  be  moved  back  and  forth  across  the  field.  (See  Ch.  IV.) 

\  49.     Spectral   or  Spectroscopic    Ocular.— (See   Micro-Spectroscope,    Ch. 

VI.)' 

DESIGNATION    OF    OCULARS 

\  50.     Equivalent   Focus. — As  with  objectives,   some  opticians  designate 
the  oculars  by  their  equivalent  focus  (2  15  ).     With  this  method  the  power  of 


No.  2 


FIG.  38.  Projection  Oculars  with  section 
removed  to  show  the  construction.  Below  are 
shown  the  upper  ends  with  graduated  circle  to 
indicate  the  amount  of  rotation  found  necessary 
to  focus  the  diaphragm  on  the  screen.  No.  2, 
No.  4.  The  numbers  indicate  the  amount  the 
ocular  magnifies  the  image  formed  by  the 
objective  as  with  the  compensation  oculars. 
(Zeiss*  Catalog.) 


the  ocular,  as  with  objectives,  varies  inversely  as  the  equivalent  focal  length, 
and  therefore  the  greater  the  equivalent  focal  length  the  less  the  magnifica- 
tion. This  seems  as  desirable  a  mode  for  oculars  as  for  objectives  and  is  com- 
ing more  and  more  into  use  by  the  most  progressive  opticians.  It  is  the 
method  of  designation  advocated  by  Dr.  R.  H.  Ward  for  many  years,  and  was 
recommended  by  the  committee  of  the  American  Microscopical  Society,  (Proc. 
Amer.  Micr.  Soc.,  1883,  p.  175,  1884,  p.  228). 

£51.  Numbering  and  Lettering.— Oculars  like  objectives  may  be  num- 
bered or  lettered  arbitrarily.  When  so  designated,  the  smaller  the  number,  or 
the  earlier  the  letter  in  the  alphabet,  the  lower  the  power  of  the  ocular. 

\  52.  Magnification. — The  compensation  oculars  and  the  Huygenian  ocu- 
lars of  some  makers  are  marked  with  the  amount  they  magnify  the  real  image. 
Thus  oculars  marked  X  4,  X  8,  indicate  that  the  real  image  of  the  objective  is 
magnified  four  or  eight  fold  by  the  ocular. 

The  projection  oculars  are  designated  simply  by  the  amount  they  multiply 
the  real  image  of  the  objective.  Thus  for  the  short  or  160  mm.  tube-length 
they  are,  X2,  X4  ;  and  for  the  long  or  250  mm.  tube,  they  are  X3  and  X6. 
That  is,  the  final  image  on  the  screen  or  the  ground  glass  of  the  photographic 
camera  will  be  2,  3,  4,  or  6  times  greater  than  it  would  be  if  no  ocular  were 
used.  See  Ch.  VIII. 


MICROSCOPE  AND  ACCESSORIES 


CH.  /] 


\  53.  Standard  Size  Oculars. — The  Royal  Microscopical  Society  of  Lon- 
don took  a  very  important  step  (Dec.  20,  1899)  in  establishing  standard  sizes 
for  oculars  and  sub-stage  condensers.  To  quote  from  the  Journal  of  the  Royal 
Microscopical  Society  for  1900,  p.  147  : 

Resolved,  "  That  the  standard  size  for  the  inside  diameter  of  the  substage 

FIG.  39.  Ocular  Screw-Micrometer 
with  compensation  ocular  6.  The  upper 
figure  shows  a  sectional  view  of  the  ocular 
and  the  screw  for  moving  the  micrometer 
at  the  right.  At  the  left  is  shown  a  clamp- 
ing sciew  to  fasten  the  ocular  to  the  upper 
part  of  the  microscope  tube.  Below  is  a 
face  view,  showing  the  graduation  on  the 
wheel.  An  ocular  micrometer  like  this  is 
in  general  like  the  cob-web  micrometer 
and  may  be  used  for  measuring  objects  of 
varying  sizes  very  accurately.  With  the 
ordinary  ocular  micrometer  very  small 
obfects  frequently  fill  but  a  part  of  an  in- 
terval of  the  micrometer,  but  with  this 
the  movable  cross  lines  traverse  the  object 
(or  rather  its  real  image)  regardless  of 
the  minuteness  of  the  object.  (Zeiss}  Cat- 
alog.} See  also  Ch.  IV. 

fitting  be  1.527  in.  =  38. 786  mm.  That  the  gauges  for  standardizing  eye-pieces 
be  the  internal  diameters  of  the  draw-tubes,  the  tightness  of  the  fit  being  left 
to  the  discretion  of  the  manufacturers." 

The  sizes  for  oculars  are  four  in  number,  i  and  2  being  most  common. 


(1)  0.9173  inch=23. 300  rum. 

(2)  1.04      inch=s6.4i6  mm. 

(3)  1.27      inch.~32.25S  mm. 

(4)  1.41      inch— 35.814  mm. 


This  is  the  Continental  size. 
This  is  the  size  used  by  the  English  opti- 
cians for  student  and  small  microscopes. 
Medium  size  binoculars  (English). 
Long  tube  binoculars. 


For  the  history  of  the   Huygenian  Ocular,  and  a  discussion  of  formulae 
for  its  construction,  see  Nelson,  J.  R.  M.  S. ,  1900,  p.  162-169. 


§  54.  Putting  an  Objective  in  Position  and  Removing  it. 
— Elevate  the  tube  of  the  microscope  by  means  of  the  coarse  adjust- 
ment, (frontispiece)  so  that  there  may  be  plenty  of  room  between 
its  lower  end  and  the  stage.  Grasp  the  objective  lightly  near  its 
lower  end  with  two  fingers  of  the  left  hand,  and  hold  it  against  the 
nut  at  the  lower  end  of  the  tube  or  the  revolving  nose  piece. 


[  CH.  I 


MICROSCOPE  AND  ACCESSORIES 


With  two  fingers  of  the  right  hand  take  hold  of  the  milled  ring 
near  the  back  or  upper  end  of  the  objective  and  screw  it  into  the 
tube  of  the  microscope  or  nose  piece.  Reverse  this  operation  for 
removing  the  objective.  By  following  this  method  the  danger  of 
dropping  the  objective  will  be  avoided. 

£  55.  Putting  an  Ocular  in  Position  and  Removing  it. — 
Elevate  the  body  of  the  microscope  with  the  coarse  adjustment  so 
that  the  objective  will  be  2  cm.  or  more  from  the  object — grasp  the 
ocular  by  the  milled  ring  next  the  eye- lens  (Fig.  37,)  and  the 
coarse  adjustment  or  the  tube  of  the  microscope  and  gently  force 
the  ocular  into  position.  In  removing  the  ocular,  reverse  the  opera- 
tion. If  the  above  precautions  are  not  taken,  and  the  oculars  fit 

FIG.  40.  Triple  nose- 
piece  or  revolver  for 
quickly  changing  objec- 
tives. 

This  covered  or  dust 
proof  form  was  original- 
ly devised  by  Winkel  of 
Goettingen;  it  is  ««<v 
furnished  by  nearly  all 
microscope  makers.  (Cut 
loaned  by  Voitgtliinder 
&  So/in,  A.  C. 

Microscope  makers  usually  construct  the  double  or  triple  nose-pieces  and 
the  length  of  the  objective  mounting  so  that  in  turning  from  one  objective  to 
another  all  will  be  approximately  in  focus.  The  objectives  arc  then  said  to  be 
par-focal. 

snugly,  there  is  danger  in  inserting  them  of  forcing  the  tube  of  the 
microscope  downward  and  the  objective  upon  the  object. 

§  56.  Putting  an  Object  Under  the  Microscope. — This  is 
so  placing  an  object  under  the  simple  microscope,  or  on  the  stage  of 
the  compound  microscope,  that  it  will  be  in  the  field  of  view  when 
the  microscope  is  in  focus  (§  57). 

With  low  powers,  it  is  not  difficult  to  get  an  object  under  the 
microscope.  The  difficulty  increases,  however,  with  the  power  of 
the  microscope  and  the  smallness  of  the  object.  It  is  usually  neces- 
sary to  move  the  object  in  various  directions  while  looking  into  the 
microscope,  in  order  to  get  it  into  the  field.  Time  is  usually  saved 


MICROSCOPE  AND  ACCESSORIES 


CH.  /] 


by  getting  the  object  in  the  center  of  the  field  with  a  low  objective 
before  putting  the  high  objective  in  position.  This  is  greatly  facili- 
tated by  using  a  nose-piece,  or  revolver.  (See  Fig.  40  and  the 
pictures  of  microscopes,  Ch.  II.) 

FIG.  41.  Krauss1  Method  of 
Marking  Objectives  on  a  Re- 
volving Nose-Piece. 

As  seen  in  the  figure,  the 
equivalent  focus  of  the  objective 
is  engraved  on  the  diaphragm 
above  the  back  lens  and  may  be 
very  readily  seen  in  rotating  the 
nose-piece.  This  is  of  great 
advantage,  as  one  can  see  what 
objective  is  coming  into  place 
without  trouble.  It  is  also  an 
advantage  in  showing  where 
each  objective  belongs  when  the 
microscope  comes  from  the 
manufacturers.  The  method 
is  coming  into  general  use. 

§  57.  Field  or  Field  of  View  of  a  Microscope. — This  is 
the  area  visible  through  a  microscope  when  it  is  in  focus.  When 
properly  lighted  and  there  is  no  object  under  the  microscope,  the 
field  appears  as  a  circle  of  light.  When  examining  an  object  it  ap- 
pears within  the  light  circle,  and  by  moving  the  object,  if  it  is  suffi- 
cient size,  different  parts  are  brought  successively  into  the  field  of 
view. 

In  general,  the  greater  the  magnification  of  the  entire  micro- 
scope, whether  the  magnification  is  produced  mainly  by  the  object- 
ive, the  ocular,  or  by  increasing  the  tube  length,  or  by  a  combina- 
tion of  all  three  (see  Ch.  IV,  under  magnification),  the  smaller  is 
the  field. 

The  size  of  the  field  is  also  dependent,  in  part,  without  regard 
to  magnification,  upon  the  size  of  the  opening  in  the  ocular  dia- 
phragm. Some  oculars,  as  the  orthoscopic  and  periscopic,  are 
so  constructed  as  to  eliminate  the  ocular  diaphragm,  and  in  conse- 
quence, although  this  is  not  the  sole  cause,  the  field  is  considerably 
increased.  The  exact  size  of  the  field  may  be  read  off  directly  by 
putting  a  stage  micrometer  under  the  microscope  and  noting  the 
number  of  spaces  required  to  measure  the  diameter  of  the  light  circle. 


CH.  /] 


MICROSCOPE  A.\H   ACCESSORIES 


33 


§  58.  The  Size  of  the  Field  of  the  microscope  as  projected 
into  the  field  of  vision  of  the  normal  human  eye  (i.  e.t  the  virtual 
image)  may  be  determined  by  the  use  of  the  camera  lucida  with  the 
drawing  surface  placed  at  the  standard  distance  of  250  millimeters 
(Ch.  IV.) 

§  59.  Table  showing  the  actual  size  in  millimeters  of  the  field  of 
a  group  of  commonly  used  objectives  and  oculars.  Compare  with  the 
graphic  representation  in  Fig.  42.  See  also  §  57. 


Kqmvalent 
Focus  and 
N.  A.  of 
Objective 

Diameter 
of  Field 
in  mm. 

Equivalent 
Focus  of 
Ocular 

Kind  of 
Ocular 

85  mm.    

15-4 
10.6 

37^  mm. 
25         " 

Huygenian 

8-3 

12^       " 

45  mm. 

7.0 
S.o 

37  yz   mm. 

2^            '  ' 

HoyzcfliaB 

4.0 

12^      " 

17  mm.    _  _  _. 

3-0 

2.0 

37X  mm. 
2t-        « 

Huygenian 

1.6 

'     12^      " 

N.  A.--  0.25 

.    5.7 

2.8 

1.4 
0.97 

180       mm. 
45 
15 

10 

Compensation 

5  mm.  _ 

0.541 

O.  371 

37^  mm. 
25        " 

Huygenian 

0.290 

12^       " 

N.  A.  =0.92 

0.850 
0.501 
0.250 
0.173 

180       mm. 
45 
15         " 

10 

Compensation 

2  mm. 

0.270 
0.186 

37>£   mm. 
25        " 

Huygenian 

0.147 

12^     '" 

N.  A.  =  i.25 

0.450 
0.251 
0.125 
0.088 

180       mm. 
45 
15 

10 

Compensation 

34  MICROSCOPE  AND  ACCESSORIES  [  CH .  I 

FUNCTION    OF    AN    OBJECTIVE 

§  60.  Put  a  50  mm.  objective  on  the  microscope  or  screw  off 
the  front  combination  of  a  16  mm.,  (^i-in),  and  put  the  back  com- 
bination on  the  microscope  for  a  low  objective. 

Place  some  printed  letters  or  figures  under  the  microscope,  and 


8  j    m  »i 

FIG.  42.  Figures  shozuing  approximately  the  actual  size  of  the  field  with 
objectives  0/85  mm.,  45  mm.,  77  mm.,  5  mm.  and  2  mm.,  equivalent  focus, 
and  an  ocular  of  37 yz  mm.  equivalent  focus  in  each  case.  This  figure  shores 
graphically  what  is  also  very  clearly  indicated  in  the  table  (_\  59}. 

light  well.  In  place  of  an  ocular  put  a  screen  of  ground  glass,  or  a 
piece  of  lens  paper,  over  the  upper  end  of  the  tube  of  the  micro- 
scope* 

Lower  the  tube  of  the  microscope  by  means  of  the  coarse  ad- 
justment until  the  objective  is  within  2  to  3  cm.  of  the  object  on  the 
stage.  Look  at  the  screen  on  the  top  of  the  tube,  holding  the  head 
about  as  far  from  it  as  for  ordinary  reading,  and  slowly  elevate  the 
tube  by  means  of  the  coarse  adjustment  until  the  image  of  the  letter 
appears  on  the  screen. 

The  image  can  be  more  clearly  seen  if  the  object  is  in  a  strong 
light  and  the  screen  in  a  moderate  light,  z.  e. ,  if  the  top  of  the  micro- 
scope is  shaded. 

The  letters  will  appear  as  if  printed  on  the  ground  glass  or  paper, 
but  will  be  inverted  (Fig.  26). 

If  the  objective  is  not  raised  sufficiently,  and  the  head  is  held 
too  near  the  microscope,  the  objective  will  act  as  a  simple  micro- 
scope. If  the  letters  are  erect,  and  appear  to  be  down  in  the  micro- 
scope and  not  on  the  screen,  hold  the  head  farther  from  it,  shade  the 


*$  61.  Ground  Glass  may  be  very  easily  prepared  by  placing  some  fine 
emery  or  carborundum  between  two  pieces  of  glass,  wetting  it  with  water  and 
then  rubbing  the  glasses  together  for  a  few  minutes.  If  the  glass  becomes  too 
opaque,  it  may  be  rendered  more  translucent  by  rubbing  some  oil  upon  it. 


CH.  /]  MICROSCOPE  AND  ACCESSORIES  35 

screen,  and  raise  the  tube  of  the  microscope  until  the  letters  do  ap- 
pear on  the  ground  glass. 

To  demonstrate  that  the  object  must  be  outside  the  principal 
focus  with  the  compound  microscope,  remove  the  screen  and  turn 
the  tube  of  the  microscope  directty  toward  the  sun.  Move  the  tube 
of  the  microscope  with  the  coarse  adjustment  until  the  burning  or 
focal  point  is  found  (§7,13).  Measure  the  distance  from  the  paper 
object  on  the  stage  to  the  objective,  and  it  will  represent  approx- 
imately the  principal  focal  distance  (Figs.  10,  n).  Replace  the 
screen  over  the  top  of  the  tube,  no  image  can  be  seen.  Slowly  raise 
the  tube  of  the  microscope  and  the  image  will  finally  appear.  If 
the  distance  between  the  object  and  the  objective  is  now  taken,  it 
will  be  found  considerably  greater  that  the  principal  focal  distance 
(compare  §  12). 

§  62  Aerial  Image. — After  seeing  the  real  image  on  the 
ground-glass,  or  paper,  use  the  lens  paper  over  about  half  of  the 
opening  of  the  tube  of  the  microscope.  Hold  the  eye  about  250 
mm.  from  the  microscope  as  before  and  shade  the  top  of  the  tube  by 
holding  the  hand  between  it  and  the  light,  or  in  some  other  way. 
The  real  image  can  be  seen  in  part  as  if  on  the  paper  and  in  part  in 
the  air.  Move  the  paper  so  that  the  image  of  half  a  letter  will  be 
on  the  paper  and  half  in  the  air.  Another  striking  experiment  is  to 
have  a  small  hole  in  the  paper  placed  over  the  center  of  the  tube 
opening,  then  if  a  printed  word  extends  entirely  across  the  diameter 
of  the  tube  its  central  part  may  be  seen  in  the  air,  the  lateral  parts 
on  the  paper.  The  advantage  of  the  paper  over  part  of  the  opening 
is  to  enable  one  to  accomodate  the  eyes  for  the  right  distance.  If 
the  paper  is  absent  the  eyes  adjust  themselves  for  the  light  circle  at 
the  back  of  the  objective,  and  the  aerial  image  appears  low  in  the 
tube.  Furthermore  it  is  more  difficult  to  see  the  aerial  image  in 
space  than  to  see  the  image  on  the  ground-glass  or  paper,  for  the  eye 
must  be  held  in  the  right  position  to  receive  the  rays  projected  from 
the  real  image,  while  the  granular  surface  of  the  glass  and  the  deli- 
cate fibres  of  the  paper  reflect  the  rays  irregularly,  so  that  the 
image  may  be  seen  at  almost  any  angle,  as  if  the  letters  were 
actually  printed  on  the  paper  or  glass. 

§  63  The  Function  of  an  Objective,  as  seen  from  these  ex- 
periments, is  to  form  an  enlarged,  inverted,  real  image  of  an  object, 


MICROSCOPE  AND  ACCESSORIES 


\_CH.  I 


this  image  being  formed  on  the  opposite  side  of  the  objective  from 
the  object  (Fig.  26). 

FUNCTION    OF  AN    OCULAR 

§  64.  Using  the  same  objective  as  for  §  53,  get  as  clear  an 
image  of  the  letters  as  possible  on  the  lens  paper  or  ground-glass 
screen.  Look  at  the  image  with  a  simple  microscope  (Fig.  19,  21) 
as  if  the  image  were  an  object. 

Observe  that  the  image  seen  through  the  simple  microscope  is 
merely  an  enlargement  of  the  one  on  the  screen,  and  that  the  letters 
remain  inverted,  that  is  they  appear  as  with  the  naked  eye  (§  12). 
Remove  the  screen  and  observe  the  aerial  image  with  the  tripod. 

Put  a  50 mm.  (A,  No.  i  or  2  in.),  ocular  i.  <?.,  an  ocular  of 
low  magnification  in  position  (§  55).  Hold  the  eye  about  10  to 
20  millimeters  from  the  eye-lens  and  look  into  the  microscope.  The 
letters  will  appear  as  when  the  simple  microscope  was  used  (see 

FIG.  43.  Diagram  of  the  simple  microscope 
showing  the  course  of  the  rays  and  all  the  images, 
and  that  the  eye  forms  an  integral  part  of  it. 

A1  jB1.  The  object  within  the  principal  focus. 
A=  B^>.  The  virtual  image  on  the  same  side  of 
the  lens  as  the  object.  It  is  indicated  by  dotted 
lines,  as  it  has  no  actual  existence. 

£2  A2.  Retinal  image  of  the  object  (A1  Bl) 
The  virtual  image  is  simply  a  projection  of  the 
retinal  image  into  the  field  of  vision. 

Axis.  The  principal  optic  axis  of  the  micro- 
scope and  of  the  eye.  Cr.  Cornea  of  the  eye.  L. 
Crystalline  lens  of  the  eye.  R.  Ideal  refracting 
surface  at  which  all  the  refractions  of  the  eye  may 
be  assumed  to  take  place. 

above),  the  image  will  become  more  distinct  by  slightly  raising    the 
tube  of  the  microscope  with  the  coarse  adjustment. 

§  65.  The  Function  of  the  Ocular,  as  seen  from  the  above, 
is  that  of  a  simple  microscope,  viz.:  It  magnifies  the  real  image 
formed  by  the  objective  as  if  that  image  were  an  object.  Compare 
the  image  formed  by  the  ocular  (Fig.  26),  and  that  formed  by  a 
simple  microscope  (Fig.  43). 


CH.  /]  MICROSCOPE  AND  ACCESSORIES  37 

It  should  be  borne  in  mind,  however,  that  the  rays  from  an 
object  as  usually  examined  with  a  simple  microscope,  extend  from 
the  object  in  all  directions,  and  no  matter  at  what  angle  the  simple 
microscope  is  held,  provided  it  is  sufficiently  near  and  points  toward 
the  object,  an  image  may  be  seen.  The  rays  from  a  real  image, 
however,  are  continued  in  certain  definite  lines  and  not  in  all  direc- 
tions; hence,  in  order  to  see  this  aerial  image  with  an  ocular  or  simple 
microscope,  or  in  order  to  see  the  aerial  image  with  the  unaided  eye, 
the  simple  microscope,  ocular  or  eye  must  be  in  the  path  of  the  rays 
(Fig.  26). 

§  66.  The  field-lens  of  a  Huygenian  ocular  makes  the  real 
image  smaller  and  consequently  increases  the  size  of  the  field;  it 
also  makes  the  image  brighter  by  contracting  the  area  of  the  real 
image.  (Fig.  36.)  Demonstrate  this  by  screwing  off  the  field-lens 
and  using  the  eye-lens  alone  as  an  ocular,  refocusing  if  necessary. 
Note  that  the  image  is  bordered  by  a  colored  haze  (§8). 

When  looking  into  the  ocular  with  the  field-lens  removed,  the 
eye  should  not  be  held  so  close  to  the  ocular,  as  the  eye-point  is  con- 
siderably farther  away  than  when  the  field-lens  is  in  place. 

§  67.  The  eye-point. — This  is  the  point  above  the  ocular  or 
simple  microscope  where  the  greatest  number  of  emerging  rays 
cross.  Seen  in  profile,  it  may  be  likened  to  the  narrowest  part  of 
an  hour  glass.  Seen  in  section  (Fig.  36),  it  is  the  smallest  and 
brightest  light  circle  above  the  ocular.  This  is  called  the  eye-point, 
for  if  the  pupil  of  the  eye  is  placed  at  this  level,  it  will  receive  the 
greatest  number  of  rays  from  the  microscope,  and  consequently  see 
the  largest  field.* 

Demonstrate  the  eye-point  by  having  in  position  an  objective 
and  ocular  as  above  (§  60).  Light  the  object  brightly,  focus  the 
microscope,  shade  the  ocular,  then  hold  some  ground-glass  or  a 
piece  of  the  lens  paper  above  the  ocular  and  slowly  raise  and  lower 
it  until  the  smallest  circle  of  light  is  found.  By  using  different 
oculars  it  will  be  seen  that  the  eye-point  is  nearer  the  eye-lens  in 
high  than  in  low  oculars,  that  is  the  eye-point  is  nearer  the  eye-lens 
for  an  ocular  of  small  equivalent  focus  than  for  one  of  greater  focal 
length. 

*  The  bright  circle  above  the  ocular  is  sometimes  called  the  Ramsden 
Circle  or  Disc.  See  Carpenter-Dallinger,  p.  106;  Spitta,  114-118;  Wright  p. 
157  ;  Beck,  p.  14. 


38  MICROSCOPE  AND  ACCESSORIES  [  CH.  I 

REFERENCES   FOR   CHAPTER    I 

In  chapter  X  will  be  given  a  bibliography,  with  full  titles,  of  the  works 
and  periodicals  referred  to. 

For  the  subjects  considered  in  this  chapter,  general  works  on  the  micro- 
scope may  be  consulted  with  great  advantage  for  different  or  more  exhaustive 
treatment.  The  most  satisfactory  work  in  English  is  Carpenter- Ballanger,  8th 
Ed.  For  the  history  of  the  microscope,  Mayall's  Cantor  Lectures  on  the 
microscope  are  very  satisfactory.  For  a  continuation  of  the  history  begun  by 
Mayall  in  the  Cantor  Lectures  see  Nelson,  Journal  of  the  Queckett  Micr.  Club, 
and  the  Jour.  Roy.  Micr.  Soc. ,  1897-1901+.  Carpenter-Dallinger,  8th  Ed. 
Petri,  Das  Mikroskop. 

The  following  special  articles  in  periodicals  may  be  examined  with  advan- 
tage: 

Apochromatic  Objectives,  etc.  Dippel  in  Zeit.  wiss.  Mikr.,  1886,  p.  303; 
also  in  the  Jour.  Roy.  Micr.  Soc.,  i886,'pp.  316,  849,  mo;  same,  1890,  p.  480, 
Zeit.  f.  Instrumentenk.,  1890,  pp.  1-6;  Micr.  Built.,  1891,  pp.  6-7. 

Tube-length,  etc.  Gage,  Proc.  Amer.  Soc.  Micrs.,  1887,  pp.  168-172;  also 
in  the  Microscope,  the  Jour.  Roy.  Micr.  Soc.,  and  in  Zeit,  wiss.  Mikr.,  1887-8. 
Bausch,  Proc.  Amer.  Soc.  Micrs.,  1890,  pp.  43-49;  also  in  the  Microscope,  1890; 
pp.  289-296. 

Aperture.  J.  D.  Cox,  Presidential  Address,  Proc.  Amer.  Soc.  Micrs.,  1884, 
pp.  5-39,  Jour.  Roy.  Micr.  Soc.,  1881,  pp.  303,  348,  365,  388;  1882,  pp.  300,  460; 
1883,  p.  790;  1884,  p.  20.  Czapski,  Theorie  der  optischen  Instrumente  nach 
Abbe. 

Theory  of  Microscopic  vision,  Wright,  Jour.  Roy.  Micr.  Soc.  1905  p.  i,. 
Biography  of  Abbe,  same,  p.  156.  See  also  the  references  to  \  40. 


CHAPTER  '  II 


LIGHTING  AND    FOCUSING  ;    MANIPULATION  OF  DRY, 

ADJUSTABLE  AND  IMMERSION  OBJECTIVES  ;  CARE 

OF   THE    MICROSCOPE    AND   OF  THE   EYES; 

LABORATORY    MICROSCOPES 


APPARATUS     AND    MATERIAL    FOR    THIS    CHAPTER 

Microscope  supplied  with  plane  and  concave  mirror,  achromatic  and  Abbe 
condensers,  dry,  adjustable  and  immersion  objectives,  oculars,*  triple  nose- 
piece.  Microscope  lamp  and  movable  condenser  (bull's  eye  or  other  form, 
Fig.  60) ;  Homogeneous  immersion  liquid,  xylene,  alcohol,  distilled  water; 
Mounted  preparation  of  fly's  wing  (§  79);  Mounted  preparation  of  Pleuro- 
sigma  (\  88,  89)  ;  Stage  or  ocular  micrometer  ($  103);  Glass  slides  and  cover- 
glasses  (Ch.  VII) ;  10  per  ct.  solution  of  salicylic  acid  in  95  per  ct.  alcohol 
(2  103);  Preparation  of  stained  bacteria  (§  119);  Vial  of  equal  parts  olive 
or  cotton  seed  oil  or  liquid  vaselin  and  xylene  ($123);  Eye  shade  (Fig.  67); 
Screen  for  whole  microscope  (Fig.  66,  68). 

FOCUSING 

|  68.  Focusing  is  mutually  arranging  an  object  and  the  microscope  so 
that  a  clear  image  may  be  seen. 

With  a  simple  microscope  (I  12)  either  the  object  or  the  microscope  or 
both  may  be  moved  in  order  to  see  the  image  clearly,  but  with  the  compound 
microscope  the  object  more  conveniently  remains  stationary  on  the  stage,  and 
the  tube  or  body  of  the  microscope  is  raised  or  lowered  (frontispiece). 

In  general,  the  higher  the  power  of  the  whole  microscope  whether  simple 
or  compound,  the  nearer  together  must  the  object  and  objective  be  brought. 
With  the  compound  microscope,  the  higher  the  objective,  and  the  longer  the 
tube  of  the  microscope,  the  nearer  together  must  the  object  and  the  objective 
be  brought.  If  the  oculars  are  not  par- focal,  the  higher  the  magnification  of 
the  ocular,  the  nearer  must  the  object  and  objective  be  brought. 

\  69.  Working  Distance.— By  this  is  meant  the  space  between  the  simple 
microscope  and  the  object,  or  between  the  front  lens  of  the  compound  micro- 
scope and  the  object,  when  the  microscope  is  in  focus.  This  working  distance 
is  always  considerably  less  than  the  equivalent  focal  length  of  the  objective. 
For  example,  the  front-lens  of  a  6  mm.  or  %  in-  objective  would  not  be  6 


40  LIGHTING  AND  FOCUSIXG  [  CH.  II 

millimeters  or  X  inch  from  the  object  when  the  microscope  is  in  focus,  but 
considerably  less  than  that  distance.  If  there  were  no  other  reason  than  the 
limited  working  distance  of  high  objectives,  it  would  be  necessary  to  use  a 
very  thin  cover-glass  over  the  object.  (See  \  27,  33.)  If  too  thick  covers  are 
used  it  may  be  impossible  to  get  an  objective  near  enough  an  object  to  get  it 
in  focus.  For  objects  that  admit  of  examination  with  high  powers  it  is  always 
better  to  use  thin  covers. 

\  70.  Free  Working  Distance  — In  the  microscope  catalog  of  Zeiss  there 
is  given  a  table  of  the  size  of  the  field  and  also  of  the  "  free  working-distance." 
This  free  working-distance  is  the  space  between  the  lower  end  of  the  objective 
and  the  cover  glass  of  Ty^  mm.  thickness,  jvhen  the  objective  is  in  focus  on  an 
object  immediately  under  the  cover.  This  is  exceedingly  practical  information 
for  a  possessor  of  a  microscope,  and  it  is  hoped  that  the  other  opticians  will 
adopt  the  suggestion.  Naturally,  however,  the  free  working-distance  for  each 
optician  should  be  reckoned  from  the  top  of  the  cover  for  which  his  unadjus- 
table  objectives  are  corrected.  If,  for  example,  the  thickness  of  cover  for 
which  an  objective  is  corrected  is  ffo  mm.  then  the  free  working-distance 
should  be  thaft  between  the  top  of  this  and  the  objective  when  the  objective  is 
in  focus  on  an  object  under  the  cover.  (See  the  table  of  cover-glass  thick- 
ness, \  33). 

LIGHTING    WITH    DAYLIGHT 

\  71.  Unmodified  sunlight  should  not  be  employed  except  in  special 
cases.  North  light  is  best  and  most  uniform.  When  the  sky  is  covered  with 
white  clouds  the  light  is  most  favorable.  To  avoid  the  shadows  produced  by 
the  hands  in  manipulating  the  mirror,  etc.,  it  is  better  to  face  the  light;  but 
to  protect  the  eyes  and  to  shade  the  stage  of  the  microscope  some  kind  of 
screen  should  be  used.  The  one  figured  in  (Fig.  66)  is  cheap  and  efficient. 
If  one  dislikes  to  face  the  window  or  lamp  it  is  better  to  sit  so  that  the  light 
will  come  from  the  left  as  in  reading. 

It  is  of  the  greatest  importance  and  advantage  for  one  who  is  to  use  the 
microscope  for  serious  work  that  he  should  comprehend  and  appreciate  thor- 
oughly the  various  methods  of  illumination,  and  the  special  appearances  due 
to  different  kinds  of  illumination. 

Depending  on  whether  the  light  illuminating  an  object  traverses  the  object 
or  is  reflected  upon  it,  and  also  whether  the  object  is  symmetrically  lighted, 
or  lighted  more  on  one  side  than  the  other,  light  used  in  microscopy  is  des- 
ignated as  reflected  and  transmitted,  axial  and  oblique. 

£  72.  Reflected,  Incident  or  Direct  Light. — By  this  is  meant  light  reflected 
upon  the  object  in  some  way  and  then  irregularly  reflected  from  the  object  to 
the  microscope.  By  this  kind  of  light  objects  are  ordinarily  seen  by  the 
unaided  eye,  and  the  objects  are  mostly  opaque.  In  Vertebrate  Histology, 
reflected  light  is  but  little  used  ;  but  in  the  study  of  opaque  objects,  like 
whole  insects,  etc.,  it  is  used  a  great  deal.  For  low  powers,  ordinary  daylight 
that  naturally  falls  upon  the  object,  or  is  reflected  or  condensed  upon  it  with  a 
mirror  or  condensing  lens,  answers  very  well.  For  high  powers  and  for 


CH.  77] 


LIGHTING  AND  FOCI 'SI AY/ 


special  purposes,  special  illuminating  apparatus  has  been  devised  (?  31).     (See 
also  Carpenter-Dallinger,  Ch   IV.) 

2  73.     Transmitted  Light. — By  this  is  meant  light  which  passes  through 
an  object  from  the  opposite  side.     The   details   of    a   photographic   negative 


44 
FIGS.  44-45.     For  full  explanation  see  Figs.  27  and  28. 


45 


are  in  many  cases  only  seen  or  best  seen  by  transmitted  light,  while  the  print 
made  from  it  is  best  seen  by  reflected  light. 

Almost  all  objects  studied  in  Vertebrate  Histology  are  lighted  by  trans- 
mitted light,  and  they  are  in  some  way  rendered  transparent  or  semi-trans- 
parent. The  light  traversing  and  serving  to  illuminate  the  object  in  working 
with  a  compound  microscope  is  usually  reflected  from  a  plane  or  concave 
mirror,  or  from  a  mirror  to  a  condenser  (  \  99),  and  thence  transmitted  to  the 
object  from  below  (Figs.  54-57). 

|  74.  —  Axial  or  Central  Light.  —  By  this  is  understood  light  reaching  the 
object,  the  rays  of  light  being  parallel  to  each  other  and  to  the  optic  axis  of 
the  microscope,  or  a  diverging  or  a  converging  cone  of  light  whose  axial  ray 
is  coincident  with  the  optic  axis  of  the  microscope.  In  either  case  the  object 
is  symmetrically  illuminated. 


2  75.  Oblique  Light.  —  This  is  ligh^  in  which  parallel  rays  from  a  plane 
mirror  form  an  angle  with  the  optic  axis  of  the  microscope  (Fig.  45).  Or  if  a 
concave  mirror  or  a  condenser  is  used,  the  light  is  oblique  when  the  axial  ray 
of  the  cone  of  light  forms  an  angle  with  the  optic  axis  (Fig.  45). 


42  LIGHTING  AND  FOCUSING  [  CH.  If 

DIAPHRAGMS 

|  76  Diaphragms  and  their  Proper  Employment. — Diaphragms  are 
opaque  disks  with  openings  of  various  sizes,  which  are  placed  between  the 
source  of  light  or  mirror  and  the  object.  In  some  cases  an  iris  diaphragm  i& 
used,  and  then  the  same  one  is  capable  of  giving  a  large  range  of  openings. 
The  object  of  a  diaphragm  in  general,  is  to  cut  off  all  adventitious  light  and 
thus  enable  one  to  light  the  object  in  such  a  way  that  the  light  finally  reach- 
ing the  microscope  shall  all  come  from  the  object  or  its  immediate  vicinity. 
The  diaphragms  of  a  condenser  serve  to  vary  its  aperture  to  the  needs  of  each 
object  and  each  objective. 

|  77.  Size  and  Position  of  Diaphragm  Opening. — When  no  condenser  is 
used  the  size  of  the  opening  in  the  diaphragm  should  be  about  that  of  the 
front  lens  of  the  objective.  For  some  objects  and  some  objectives  this  rule 
may  be  quite  widely  departed  from  ;  one  must  learn  by  trial. 

When  lighting  with  a  mirror  the  diaphragm  should  be  as  close  as  possible 
to  the  object  in  order,  (a)  that  it  may  exclude  all  adventitious  light  from  the 
object;  (b)  that  it  may  not  interfere  with  the  most  efficient  illumination  from 
the  mirror  by  cutting  off  a  part  of  the  illuminating  pencil.  If  the  diaphragm 
is  a  considerable  distance  below  the  object,  (i )  it  allows  considerable  adventi- 
tious light  to  reach  the  object  and  thus  injures  the  distinctness  of  the  micro- 
scope image;  (2)  it  prevents  the  use  of  very  oblique  light  unless  it  swings 
with  the  mirror  ;  (3)  it  cuts  off  a  part  of  the  illuminating  cone  from  a  concave 
mirror.  On  the  other  hand,  even  with  a  small  diaphragm,  the  whole  field 
will  be  lighted. 

With  an  illuminator  or  condenser  (Figs.  47,  54),  the  diaphragm  serves  to- 
narrow  the  pencil  to  be  transmitted  through  the  condenser,  and  thus  to  limit 
the  aperture  (see  §95).  Furthermore,  by  making  the  diaphragm  opening 
eccentric,  oblique  light  may  be  used,  or  by  using  a  diaphragm  with  a  slit 
around  the  edge  (central  stop  diaphragm),  the  center  remaining  opaque,  the 
object  may  be  lighted  with  a  hollow  cone  of  light,  all  of  the  rays  having  great 
obliquity.  In  this  way  the  so-called  dark-ground  illumination  may  be  pro- 
produced  (I  103;  Fig.  57). 

ARTIFICIAL    ILLUMINATION 

§  78.  For  evening  work  and  for  certain  special  purposes,  artificial  illumi- 
nation is  employed.  A  good  petroleum  (kerosene)  lamp  with  flat  wick  has 
been  found  very  satisfactory,  also  an  incandescent  electric  or  Welsbach  light, 
but  for  brilliancy  and  for  the  actinic  power  necessary  for  very  rapid  photo- 
micrography (seeCh.  VIII)  the  electric  arc  lamp  cr  an  acetylene  lamp  serves 
well.  Whatever  source  of  artificial  light  is  employed,  the  light  should  be 

brilliant  and  steady. 

• 

LIGHTING    EXPERIMENTS 

§  79.     Lighting  with  a  Mirror. — As  the  following  experi- 


CH.  //]  LIGHTING  AND  FOCUSING  43 

ments  are  for  mirror  lighting  only,  remove  the  subtage  condenser 
if  present  (see  §  90,  for  condenser).  Place  a  mounted  fly's  wing 
under  the  microscope,  put  the  i6mm.  (^iin.)  or  other  low  objec- 
tive in  position,  also  a  low  ocular.  With  the  coarse  adjustment 
lower  the  tube  of  the  microscope  to  within  about  i  cm.  of  the  object. 
Use  an  opening  in  the  diaphragm  about  as  large  as  the  front  lens 
of  the  objective;  then  with  the  plane  mirror  try  to  reflect  light  up 
through  the  diaphragm  upon  the  object.  One  can  tell  when  the 
field  (§  57)  is  illuminated,  by  looking  at  the  object  on  the  stage, 
but  more  satisfactorily  by  looking  into  the  microscope.  It  some- 
times requires  considerable  manipulation  to  light  the  field  well. 
After  using  the  plane  side  of  the  mirror  turn  the  concave  side  into 
position  and  light  the  field  with  it.  As  the  concave  mirror  con- 
denses the  light,  the  field  will  look  brighter  with  it  than  with  the 
plane  mirror.  It  is  especially  desirable  to  remember  that  the  excel- 
lence of  lighting  depends  in  part  on  the  position  of  the  diaphragm 
(§  7?)-  ^  tne  greatest  illumination  is  to  be  obtained  from  the  con- 
cave mirror,  its  position  must  be  such  that  its  focus  will  be  at  the 
level  of  the  object.  This  distance  can  be  very  easily  determined  by 
finding  the  focal  point  of  the  mirror  in  full  sunlight. 

§  So.  Use  of  the  Plane  and  of  the  Concave  Mirror. — The 
mirror  should  be  freely  movable,  and  have  a  plane  and  a  concave 
face.  The  concaved  face  is  used  when  a  large  amount  ot  light  is 
needed,  the  plane  face  when  a  moderate  amount  is  needed  or  when 
it  is  necesssay  to  have  parallel  rays  or  to  know  the  direction  of  the 
rays. 

FOCUSING   EXPERIMENTS* 
§  8r.     Focusing  with  Low  Objectives. — Place   a    mounted 


\  82.  *Par-Focal  Oculars. — By  this  is  meant  oculars  of  different  power 
in  which  the  microscope  remains  in  focus  on  changing  the  oculars. 

As  originally  constructed  the  microscope  had  to  be  focused  every  time  the 
oculars  were  changed.  Mr.  Edward  Pennock  in  seeking  to  overcome  this 
inconvenience  wrote  to  Professor  Abbe  for  advice  in  188 r.  After  successfully 
producing  oculars  of  different  powers  for  the  Acme  microscopes  of  Jas.  W. 
Queen  &  Co.,  according  to  the  directions  given  by  Professor  Abbe,  Mr.  Pen- 
nock  as  editor  of  the  Microscopical  Bulletin  and  Science  News  published  in 
Vol.  Ill,  1886,  pp.  9-10,  the  following  with  Professor  Abbe's  letter  :  "  Chang- 
ing Eyepieces  without  altering  focus,  etc.  Some  years  ago  the  writer  in 
looking  up  certain  questions  in  connection  with  eyepieces  took  occasion  to 


44  LIGHTING  AND  FOCL7SING  \_CH.1I 

fly's  wing  under  the  microscope  ;  put  the  16  mm.  (^  in.)  objective 
in  position,  and  also  the  lowest  ocular.  Select  the  proper  opening 
in  the  diaphragm  and  light  the  object  well  with  transmitted  light 

(§  73-  77)- 

Hold  the  head  at  about  the  level  of  the  stage,  look  toward  the 
window,  and  between  the  object  and  the  front  of  the  objective  ;  with 
the  coarse  adjustment  lower  the  tube  until  the  objective  is  within 
about  half  a  centimeter  of  the  object.  Then  look  into  the  micro- 
scope and  slowly  elevate  the  tube  with  the  coarse  adjustment.  The 
image  will  appear  dimly  at  first,  but  will  become  very  distinct  by 
raising  the  tube  still  higher.  If  the  tube  is  raised  too  high  the 
image  will  become  indistinct,  and  finally  disappear.  It  will  again 
appear  if  the  tube  is  lowered  the  proper  distance. 

When  the  microscope  is  well  focused  try  both  the  concave  and 
the  plane  mirrors  in  various  positions  and  note  the  effect.  Put  a 
high  ocular  in  place  of  the  low  one  (§  50).  If  the  oculars  are  not 
par-focal  it  will  be  necessary  to  lower  the  tube  somewhat  to  get  the 
microscope  in  focus. 

Pull  out  the  draw-tube  4  to  6  cm.,  thus  lengthening  the  body  of 
the  microscope  ;  it  will  be  found  necessary  to  lower  the  tube  of  the 
microscope  somewhat.  (For  reason,  see  Fig.  65.) 

§  83.  Pushing  in  the  Draw-Tube. — To  push  in  the  draw- 
tube,  grasp  the  large  milled  ring  of  the  ocular  with  one  hand,  and 
the  milled  head  of  the  coarse  adjustment  with  the  other,  and  grad- 


write  to  Professor  Abbe,  and  his  reply,  kindly  given,  is  so  clear  and  to  the 
point,  and  of  such  interest  and  value,  that  we  take  the  liberty  of  publishing  it 
for  the  benefit  of  our  readers." 

"Jena,  June  25th,  1881.  Dear  Sir  :  The  question  which  you  ask  admits  of  a 
simple  answer  :  In  order  to  change  the  oculars  of  a  microscope  without  chang- 
ing the  focus  of  the  objective,  neither  the  diaphragm  nor  the  field  lens  must 
come  to  the  same  place  in  the  microscope  tube,  but  the  anterior  (lower)  focal 
points  of  the  ocular  systems  must  do  this.  In  the  case  of  a  Huygehenian 
eyepiece,  the  said  anterior  focus  is  a  virtual  one  situated  above  the  field  lens 
at  a  place  D*,  which  is  more  distant  from  the  field  lens  than  the  diaphragm  D. 
The  level  of  D*  is  the  place  where  the  virtual  image  of  the  diaphragm  appears 
to  an  observer  looking  through  the  field  lens.  Rays  which  are  required  to 
emerge  from  the  eye  lens  as  parallel  rays  (or  nearly  parallel)  must  of  course 
enter  into  the  ocular  converging  to  the  point  D*.  Consequently  if  different 
oculars  are  inserted  successively  in  such  a  way  that  the  point  D*  comes  to  the 
same  place  of  the  tube  always,  the  conjugate  foci  of  object  and  image  in  the 
objective  remain  unaltered." 


CH.  //] 


LIGHTING  AND  FOCUSL\(, 


45 


ually  push  the  draw-tube  into  the  tube.  If  this  were  done  without 
these  precautions  the  objective  might  be  forced  against  the  object 
and  the  ocular  thrown  out  by  the  compressed  air. 

§  84.  Focusing  with  High  Objectives. — Employ  the  same 
object  as  before,  elevate  the  tube  of  the  microscope  and,  if  no  revolv- 
ing nose-piece  is  present,  remove  the  16  mm.  (~ $  in.)  objective  as 
indicated.  Put  a  4  or  3  mm.  ('£  or  '  in.)  or  a  higher  objective  in 
place,  and  use  a  low  ocular. 


— ttfi ~*TET 

_  fj^TT' 


FUW  Uuf 


FIG.  46 

"  This  arrangement  and  no  other  one  fulfills  at  the  same  time  the  other 
request  that  the  amplification  of  the  microscope  with  different  oculars  should 
be  in  exact  inverse  proportion  of  the  equivalent  focal  length  of  the  oculars." 

"  The  position  of  the  point  D*  may  be  easily  calculated  for  every  ocular. 
If  A  is  the  distance  of  the  diaphragm  from  the  field  lens  and  X  the  focal  length 
of  that  lens,  the  distance  of  the  focus  D*  above  the  diaphragm  (z.  e.  the  dis- 

A2 

tance  from  D  to   D*)  will  be:     /j— .     Hoping  that  these  explanations 

X— A 
will  be  found  satisfactory  for  your  aim,  I  remain  yours  sincerely, 

DR.  E.  ABBE." 

On  p.  31  of  the  Bulletin  is  the  following  :  "  Par- focal  Eye-pieces.  Referring 
to  the  article  in  the  April  issue  of  the  Bulletin,  on  changing  ej-e-pieces  with- 
out altering  focus,  etc.,  we  announce  that  we  are  prepared  to  furnish  eye- 
pieces as  there  described  with  our  Acme  microscopes  at  a  slight  additional 
expense. 

We  have  named  these  eye-pieces  PAR-FOCAL,  meaning  of  equal  focus, 
from  the  Latin  par  (equal)  and  focus." 


46  LIGHTING  AND  FOCUSING  [  CH.  II 

Light  well,  and  employ  the  proper  opening  in  the  diaphragm, 
etc.  (§  77.)  Look  between  the  front  of  the  objective  and  the  ob- 
ject as  before  (§  81),  and  lower  the  tube  with  the  coarse  adjustment 
till  the  objective  almost  touches  the  cover-glass  over  the  object. 
Look  into  the  microscope,  and  with  the  coarse  adjustment,  raise  the 
tube  very  slowly  until  the  image  begins  to  appear,  then  turn  the 
milled  head  of  the  fine  adjustment  (frontispiece),  first  one  way  and 
then  the  other,  if  necessary,  until  the  image  is  sharply  defined. 

In  practice  it  is  found  of  great  advantage  to  move  the  prepara- 
tion slightly  while  focusing.  This  enables  one  to  determine  the 
approach  to  the  focal  point  either  from  the  shadow  or  the  color,  if 
the  object  is  colored.  With  high  powers  and  scattered  objects  there 
might  be  no  object  in  the  small  field  (see  §  57  Fig.  42  for  size  of 
field).  By  moving  the  preparation  an  object  will  be  moved  across 
the  field  and  its  shadow  gives  one  the  hint  that  the  objective  is  ap- 
proaching the  focal  point.  It  is  sometimes  desirable  to  focus  on  the 
edge  of  the  cement  ring  or  on  the  little  ring  made  by  the  marker 
(see  Figs.  70-75.) 

Note  that  this  high  objective  must  be  brought  nearer  the  ob- 
ject than  the  low  one,  and  that  by  changing  to  a  higher  ocular  (if 
the  oculars  are  not  par-focal)  or  lengthening  the  tube  of  the  micro- 
scope it  will  be  found  necessary  to  bring  the  objective  still  nearer 
the  object,  as  with  the  low  objective.  (For  reason  see  Fig.  65.) 

§  86.  Always  Focus  Up,  as  directed  above.  If  one  lowers 
the  tube  only  when  looking  at  the  end  of  the  objective  as  directed 


|  85.  Par-Focal  Objectives. — By  this  is  meant  that  the  objectives  are  so 
mounted  that  when  changed  on  the  microscope  the  object  will  remain  approx- 
imately in  focus  for  all  if  it  is  in  focus  for  any  one.  The  expression  is  appli- 
cable especially  to  a  group  of  objectives  on  a  revolving  nose-piece.  The 
tube-length  of  the  microscope  must  remain  constant,  for  only  a  slight  change 
in  length  (10  to  15  mm.)  will  destroy  the  parfocalization.  In  case  the 
objectives  on  a  revolving  nose-piece  are  somewhat  out  of  parfocalizatian  one 
may  correct  it  by  getting  one  in  exact  focus,  and  then  noting  when  the  others 
are  rotated  in  place  whether  the  microscope  must  be  focused  up  or  down  to 
bring  the  objective  in  focus. 

If  one  winds  a  piece  of  string  around  the  objective  that  is  up  too  high  it 
will  prevent  it  entering  the  nut  of  the  nose-piece  so  far  and  hold  it  down  at  the 
right  level. 

It  is  not  known  by  the  writer  who  first  thought  of  arranging  the  objectives 
so  that  the  different  powers  would  be  in  focus  when  in  position.  It  is  a  recent 
improvement,  coming  in  as  a  necessary  consequence  of  parfocalizing  the  oculars. 


CH.  77]  LIGHTING  AND  FOCUSING  47 

above,  there  will  be  no  danger  of  bringing  the  objective  in  contact 
with  the  object,  as  may  be  done  if  one  looks  into  the  microscope 
and  focuses  down. 

When  the  instrument  is  well  focused,  move  the  object  around 
in  order  to  bring  different  parts  into  the  field.  It  may  be  necessary 
to  re- focus  with  the  fine  adjustment  every  time  a  different  part  is 
brought  into  the  field.  In  practical  work  one  hand  is  kept  on  the 
fine  adjustment  constantly,  and  the  focus  is  continually  varied. 

§  87.  Determination  of  Working  Distance.  As  stated  in 
§  69,  this  is  the  distance  between  the  front  lens  of  the  objective  and 
the  object  when  the  objective  is  in  focus.  It  is  always  less  than  the 
equivelent  focal  length  of  the  objective. 

Make  a  wooden  wedge  10  cm.  long  which  shall  be  exceedingly 
thin  at  one  end  and  about  20  mm.  thick  at  the  other.  Place  a  slide 
on  the  stage  and  some  dust  on  the  slide.  Do  not  use  a  cover-glass. 
Focus  the  dust  carefully  first  with  the  low  then  with  the  high  ob- 
jective. When  the  objective  is  in  focus  push  the  wedge  under  the 
objective  on  the  slide  untifit  touches  the  objective.  Mark  the  place 
of  contact  with  a  pencil  and  then  measure  the  thickness  of  the 
wedge  with  a  rule  opposite  the  point  of  contact.  This  thickness 
will  represent  very  closely  the  working  distance.  For  measuring 
the  thickness  of  the  wedge  at  the  point  of  contact  for  the  high  ob- 
jective use  a  steel  scale  ruled  in  4-  mm.  and  the  tripod  to  see  the  di- 
visions. Or  one  may  use  a  cover-glass  measure  (Ch.  VIII)  for  de- 
termining the  thickness  of  the  wedge. 

For  the  higher  powers  if  one  has  a  microscope  in  which  the  fine 
adjustment  is  graduated,  the  working  distance  may  be  readily  de- 
termined when  the  thickness  of  the  cover-glass  over  the  specimen 
is  known,  as  follows  :  Get  the  object  in  focus,  lower  the  tube  of 
the  microscope,  until  the  front  of  the  objective  just  touches  the 
cover-glass.  Note  the  position  of  the  micrometer  screw  and  slowly 
focus  up  with  the  fine  adjustment  until  the  object  js  in  focus.  The 
distance  the  objective  was  raised  plus  the  thickness  of  the  cover- 
glass  represents  the  working  distance.  For  example,  a  3  mm.  ob- 
jective after  being  brought  in  contact  with  the  cover-glass  was 
raised  by  the  fine  adjustment  a  distance  represented  by  16  of  the 
divisions  on  the  head  of  the  micrometer  screw.  Each  division  rep- 
resented o.oi  mm.,  consequently  the  objective  was  raised  o.  16  mm. 


48  LIGHTING  AND  FOCUSING  [  CH.  11 

As  the  cover- glass  on   the  specimen  used  was  0.15  mm.  the   total 
working  distance  is  0.16-1-0.15=0.31  mm. 

CENTRAL    AND    OBLIQUE    LIGHT    WITH    A    MIRROR 

§  88.  Axial  or  Central  Light  (§  74). — Remove  the  con- 
denser or  any  diaphragm  from  the  substage,  then  place  a  preparation 
containing  minute  air  bubbles  under  the  microscope.  The  prepara- 
tion may  be  easily  made  by  beating  a  drop  of  mucilage  on  the  slide 
and  covering  it  (see  Ch.  III).  Use  a  4  or  3  mm.,  (^in.)  or  No.  7 
objective  and  a  medium  ocular.  Focus  the  microscope  and  select  a 
very  small  bubble,  one  whose  image  appears  about  i  mm.  in  diameter, 
then  arrange  the  plane  mirror  so  that  the  light  spot  in  the  bubble 
appears  exactly  in  the  center.  Without  changing  the  position  of 
the  mirror  in  the  least,  replace  the  air  bubble  preparation  by  one  of 
Pleurosigma  angulatum  or  some  other  finely  marked  diatom.  Study 
the  appearance  very  carefully. 

§  89.  Oblique  Light  (§  75).— Swing  the  mirror  far  to  one 
side  so  that  the  rays  reaching  the  object  may  be  very  oblique  to  the 
optic  axis  of  the  microscope.  Study  carefully  the  appearance  of  the 
diatom  with  the  oblique  light.  Compare  the  appearance  with  that 
where  central  light  is  used.  The  effect  of  oblique  light  is  not  so 
striking  with  histological  preparations  as  with  diatoms. 

It  should  be  especially  noted  in  §§  88,  89,  that  one  cannot 
determine  the  exact  direction  of  the  rays  by  the  position  of  the  mir- 
ror. This  is  especially  true  for  axial  light  (§  88).  To  be  certain 
the  light  is  axial  some  such  test  as  that  given  in  §  88  should  be 
applied.  (See  also  Ch.  Ill,  under  Air-bubbles.) 

CONDENSERS  OR  ILLUMINATORS* 

§  90.     These  are  lenses   or   lens-systems    for   the    purpose   of 


*No  one  has  stated  more  clearly,  or  appreciated  more  truly  the  value  of 
correct  illumination  and  the  methods  of  obtaining  it,  than  Sir  David  Brewster, 
1820,  1831.  He  says  of  illumination  in  general:  "The  art  of  illuminating 
microscopic  objects  is  not  of  less  importance  than  that  of  preparing  them  for 
observation."  "The  eye  should  be  protected  from  all  extraneous  light,  and 
should  not  receive  any  of  the  light  which  proceeds  from  the  illuminating 
center,  excepting  that  portion  of  it  which  is  transmitted  through  or  reflected 
from  the  object."  So  likewise  the  value  and  character  of  the  substage  con- 


C //.//]  LIGHTING  AND  FOCUSING  49 

illuminating  with  transmitted  light  the  object  to  be  studied  with 
the  microscope. 

For  the  highest  kind  of  investigation  their  value  cannot  be 
over-estimated.  They  may  be  used  either  with  natural  or  artificial 
light,  and  should  be  of  sufficient  numerical  aperture  to  satisfy 
objectives  of  the  widest  angle. 

It  is  of  the  greatest  advantage  to  have  the  sub-stage  condenser 
mounted  so  that  it  may  be  easily  moved  up  or  down  under  the  stage. 
The  iris  diaphragm  is  so  convenient  that  it  should  be  furnished  in 
all  cases,  and  there  should  be  marks  indicating  the  N.  A.  (§  36)  of  the 
condenser  utilized  with  different  openings.  Finally  the  condenser 
should  be  supplied  with  central  stops  for  dark-ground  illumination 
(§  103)  and  with  blue  and  neutral  tint  glasses  to  soften  the  glare 
when  artificial  light  is  used  (§  100,  104). 

Condensers  or  Illuminators  fall  into  two  great  groups,  the 
Achromatic,  giving  a  large  aplanatic  cone,  and  Non-achromatic, 
giving  much  light,  but  a  relatively  small  aplanatic  cone  of  light. 

£91.  Achromatic  Condenser. — It  is  still  believed  by  all  ex- 
pert microscopists  that  the  contention  of  Brewster  was  right,  and 
the  condenser  to  give  the  greatest  aid  in  elucidating  microscopic 
structure  must  approach  in  excellence  the  best  objectives.  That  is, 
it  should  be  as  free  as  possible  from  spherical  and  chromatic  aberra- 
tion, and  therefore  would  transmit  to  the  object  a  very  large  aplan- 
atic cone  of  light.  Such  condensers  are  especially  recommended 
for  photo-micrography  by  all,  and  those  who  believe  in  getting  the 
best  possible  image  in  every  case  are  equally  strenuous  that  achro- 
matic condensers  should  be  used  for  all  work.  Unfortunately  good 
condensers  like  good  objectives  are  expensive,  and  student  micro- 
scopes as  well  as  many  others  are  usually  supplied  with  the  non- 
achromatic  condensers  or  with  none. 

Many  excellent  achromatic  condensers  have  been  made,  but  the 


denser  was  thoroughly  understood  and  pointed  out  by  him  as  follows:  "I 
have  no  hesitation  in  saying  that  the  apparatus  for  illumination  requires  to  be 
as  perfect  as  the  apparatus  for  vision,  and  on  this  account  I  would  recommend 
that  the  illuminating  lens  should  be  perfectly  free  of  chromatic  and  spherical 
aberration,  and  the  greatest  care  be  taken  to  exclude  all  extraneous  light  both 
from  the  object  and  from  the  eye  of  the  observer."  See  Sir  David  Brewster's 
treatise  on  the  Microscope,  1837,  pp.  136,  138,  146,  and  the  Edinburgh  Journal 
of  Science,  new  series,  No.  n  (1831)  p.  83. 


50  LIGHTING  AND  FOCUSING  [  CH.  II 

most  perfect  of  all  seems  to  be  the  apochromatic  of  Powell  and  Lea- 
land  (Carpenter-Dallinger,  p.  302).  To  attain  the  best  that  was 
possible  many  workers  have  adopted  the  plan  of  using  objectives  as 
condensers.  A  special  substage  fitting  is  provided  with  the  proper 
screw  and  the  objective  is  put  into  position,  the  front  lens  being 
next  the  object.  As  will  be  seen  below  (§94,  95),  the  full  aperture 
of  an  objective  can  rarely  be  used,  and  for  histological  preparations 
perhaps  never,  so  that  an  objective  of  greater  equivalent  focus,  i  e., 
lower  power,  is  used  for  the  condenser  than  the  one  on  the  micro- 
scope. It  is  much  more  convenient,  however,  to  have  a  special 
condenser  with  iris  diaphragm  or  special  diaphragms  so  that  one 
may  use  any  aperture  at  will,  and  thus  satisfy  the  conditions  neces- 
sary for  lighting  different  objects  for  the  sa'me  objective  and  for 
lighting  with  objectives  of  different  apertures.  An  excellent  con- 
denser of  this  form  has  been  produced  by  Zeiss  (Fig.  47).  It  has  a 
total  numerical  aperture  of  i.oo,  and  an  aplanatic  aperture  of  0.65. 

FiG.  47.  Zeiss'  Achromatic  Con- 
denser, c.  s.  c.  s.  Centering  screws 
for  changing  the  position  of  the  con- 
denser and  making  its  axis  continuous 
with  that  of  the  microscope.  A  seg- 
ment of  the  condenser  is  cut  away  to 
show  the  combination  of  lenses.  For 
very  low  powers  the  upper  lens  is 
sometimes  screwed  off.  There  is  an 
iris  diaphragm  between  the  middle 
and  lower  combinations.  (Zeiss' 
Catalog, ) 

§  92.  Centering  the  Condenser. — To  get  the  best  possible 
illumination  for  bringing  out  in  the  clearest  manner  the  minute  de- 
tails of  a  microscopic  object  two  conditions  are  necessary,  viz. :  The 
principal  optic  axis  of  the  condenser  must  be  continuous  with  that 
of  the  microscope  (see  frontispiece)  and  the  object  must  be  in  the 
focus  of  the  condenser,  i.e. ,  at  the  apex  of  the  cone  of  light  given 
by  the  condenser. 

The  centering  is  most  conveniently  accomplished  as  follows  al- 
though daylight  may  be  used  with  almost  equal  facility.  A  very 
small  diaphragm  is  put  below  the  condenser.  (If  the  Zeiss  achro- 
matic condenser  is  used,  the  diaphragm  of  the  Abbe  illuminator 
serves  for  this.  If  there  is  no  pin-hole  diaphragm  one  can  be  made 


Cll.  //]  LIGHTING  AND  FOCUS/. \<;  51 

of  stiff,  black  paper.  Care  must  be  taken,  however,  to  make  the 
opening  exactly  central.  This  is  best  accomplished  by  putting  the 
paper  disc  over  the  iris  or  metal  diaphragms  and  then  making  the  hole 
in  the  center  of  the  small  circle  uncovered  by  the  metal  diaphragm 
For  the  hole  a  fine  needle  is  best).  Light  well  and  lower  the  objec- 
tive so  that  it  is  at  about  its  working-distance  from  the  top  of  the 
condenser.  If  now  the  condenser  is  lowered  or  racked  away  from 
the  objective  the  image  of  the  diaphragm  will  appear.  If  the  open- 
ing is  not  central  it  should  be  made  so  by  using  the  centering  screws 
of  the  condenser. 

A  better  plan  than  to  lower  the  condenser  to  focus  the  image  of 
the  diaphragm,  is  to  raise  the  body  of  the  microscope  slowly  with 
the  coarse  adjustment.  It  is  almost  impossible  to  make  apparatus 
so  accurate  that  two  parts  like  the  body  of  the  microscope  and  the 
substage,  each  working  on  different  sliding  surfaces,  shall  continue 
in  exactly  the  same  plane.  So  one  will  find  that  if  the  condenser  be 
accurately  centered  with  the  condenser  lowered,  and  then  the  con- 
denser be  racked  up  close  to  the  stage  and  the  image  of  the  dia- 
phragm opening  brought  again  into  focus  by  racking  up  the  body  of 
the  microscope,  it  will  not  be  accurately  centered  in  most  cases. 
For  this  reason  it  is  advised  that  the  condenser  be  left  in  position 
close  to  the  stage  and  the  tube  of  the  microscope  be  used  to  focus 
the  diaphragm  exactly  as  in  ordinary  work. 

FIG.  48.  Short's  that  the  optic 
axis'  of  the  condenser  docs  not  coin- 
cide with  that  of  the  microscope.  (D). 
Image  of  the  diaphragm  of  the  con- 
denser s/iozai  at  one  side  of  the  field 
of  view. 

FIG.  49.  Shores  the  image  of  the 
diaphragm  (£))  in  the  center  of  the 
field  of  the  microscope,  and  thus  the 
coincidence  of  the  axis  of  the  con- 
denser n'ith  that  of  the  microscope. 

§  93.  Centering  the  Image  of  the  Source  of  Illumination. — 
For  the  best  results  it  is  not  only  necessary  that  the  condenser  be 
properly  centered,  but  that  the  object  to  be  studied  should  be  in  the 
image  of  the  source  of  illumination  and  that  this  should  also  be  cen- 
tered (Figs.  50,  51).  After  the  condenser  itself  is  centered  the  iris 
diaphragm  is  opened  to  its  full  extent  or  the  diaphragm  carrier 


LIGHTING  AND  FOCUS  INC, 


\_CH. 


turned  wholly  aside.  A  transparent  specimen  like  the  fly's  wing  is 
put  under  the  microscope  and  focused.  The  condenser  is  then 
turned  up  and  down  until  the  image  of  the  flame  is  apparently  on 
the  specimen.  If  this  cannot  be  accomplished  the  relative  position 
of  the  lamp  and  condenser  is  not  correct  and  should  be  so  changed 
that  the  image  of  the  edge  of  the  flame  is  sharply  defined.  This 
image  must  also  be  centered.  This  is  easily  accomplished  by  manip- 
ulation of  the  mirror  or,  if  a  lamp  is  used,  by  changing  the  position 
of  the  lamp  or  of  the  bull's  eye  (Fig.  60). 

§  94.  Proper  Numerical  Aperture  of  the  Condenser. — As 
stated  above,  the  aperture  of  the  condenser  should  have  a  range  by 
means  of  properly  selected  diaphragms  to  meet  the  requirements  of 
all  objectives  from  the  lowest  to  those  of  the  highest  aperture.  It  is 
found  in  practice  that  for  diatoms,  etc.,  the  best  images  are  obtained 
when  the  object  is  lighted  with  a  cone  which  fills  about  three-fourths 
of  the  diameter  of  the  back  lens  of  the  objective  with  light,  but  for 
histological  and  other  preparations  of  lower  refractive  power  only 
one-half  or  one-third  the  aperture  often  gives  the  most  satisfactory 
images  (§  4°)- 

FIG.  50.  Shows  the  image  of 
the  flame  (Fl.)  in  the  center  (C) 
of  the  field  of  the  microscope  and 
illuminating  the  object. 

FIG.  51.  Shows  the  image  of 
the  flame  (Fl.)  at  one  side  of  the 
center  ( Exc. )  and  not  properly  il- 
luminating the  object. 


Exc 


FIG.  50 


FIG.  51 


To  determine  this  in  any  case  focus  upon  some  very  transparent 
object,  take  out  the  ocular,  look  down  the  tube  at  the  back  lens.  If 
less  than  three-fourths  of  the  back  lens  is  lighted,  increase  the  open- 
ing in  the  diaphragm — if  more  than  three-fourths  diminish  it.  For 
some  objects  it  is  advantageous  to  use  less  than  three-fourths  of  the 
aperture.  Experience  will  teach  the  best  lighting  for  special  cases. 

§  95.  Aperture  of  the  Illuminating  Cone  and  the  Field.— 
It  is  to  be  remarked  that  with  a  very  small  source  of  light  the  entire 
aperture  of  the  objective  may  be  filled  if  a  proper  illuminator  or 
condenser  is  used.  The  aperture  depends  on  the  diaphragm  used 


CH.  //]  LIGHTING  AND  FOCUSIXC  53 

with  the  condenser.  And  the  size  of  the  diaphragm  must  be 
directly  as  the  aperture  of  the  objective.  That  is,  it  is  just  the 
reverse  of  the  rule  for  diaphragms  where  no  condenser  is  used 
(§  76) ;  for  there  the  diaphragm  is  made  large  for  low  powers,  and 
consequently  low  apertures,  while  with  the  condenser  the  diaphragm 
is  made  small  for  low  and  large  for  high  powers  as  the  aperture  is 


Obj 


o 


fllnm  Ilium 

FIG.  5?  FIG.  53 

FIGS.  52-53.  Figures  showing  the  dependence  of  the  objective  upon  the 
illuminating  cone  of  the  condenser  (Nelson). 

FIG.  52  (A).  The  illuminating  cone  from  the  condenser  (Ilium.).  This 
is  seen  to  be  just  sufficient  to  fill  the  objective  (Obj. ). 

(B. )  The  back  lens  of  the  objective  entirely  filled  with  light,  showing  that 
the  numerical  aperture  of  the  illuminator  is  equal  to  that  of  the  objective. 

FIG.  53  (A).  In  this  figure  the  illuminating  cone  from  the  condenser 
( Ilium. )  is  seen  to  be  sufficient  to  fill  the  objective  (Obj.) . 

(B.)  The  back  lens  of  the  objective  only  partly  filled  with  light,  due  to  the 
restricted  aperture  of  the  illuminator. 

greater  in  the  high  powers  of  a  given  series  of  objectives.  It  is 
very  instructive  to  demonstrate  this  by  using  a  16  mm.  objective 
and  opening  the  diaphragm  of  the  condenser  till  the  back  lens  is 
just  filled  with  light.  Then  if  one  uses  a  3  or  4  mm.  objective  it 
will  be  seen  that  the  back  lens  of  the  higher  objective  is  only  partly 
filled  with  light  and  to  fill  it  the  diaphragm  must  be  much  more 
widely  opened. 

With  a  condenser,  then,  the  diaphragm  has  simply  to  regulate 
the  aperture  of  the  illuminating  cone,  and  has  nothing  to  do  with 
lighting  a  large  or  a  small  field. 

With  the  condenser  there  are  two  conditions  that  must  be  ful- 
filled,— the  proper  aperture  must  be  used,  and  that  is  determined 
by  the  diaphragm,  and  secondly  the  whole  field  must  be  lighted. 
The  latter  is  accomplished  by  using  a  larger  source  of  light,  as  the 
face  instead  of  the  edge  of  a  lamp  flame,  or  by  lowering  or  raising 


54  LIGHTING  AND  FOCI  'SIXG  [  CH.  II 

the  condenser  so  that  the  object  is  not  in  the  focus  of  the  condenser, 
but  above  or  below  it,  and  therefore  lighted  by  a  converging  or 
diverging  beam  where  the  light  is  spread  over  a  greater  area  (Figs. 
54-57.  §  99)- 

§  96.  Non-Achromatic  Condensers. — Of  the  non-achromatic 
condensers  or  illuminators,  the  Abbe  condenser  or  illuminator  is  the 
one  most  generally  used.  From  its  cheapness  it  is  also  much  more 
commonly  used  than  the  achromatic  condenser.  It  consists  of  two 
or  three  very  large  lenses  and  transmits  a  cone  of  light  of  1.20  N.A. 
to  1.40  N.A.,  Figs.  58-59,  but  the  aberrations,  both  spherical  and 
chromatic,  are  very  great  in  both  forms.  Indeed,  so  great  are  they 
that  in  the  best  form  with  three  lenses  and  an  illuminating  cone  of  i .  40 
N.  A.,  the  aplanatic  cone  transmitted  is  only  0.5,  and  it  is  the  apla- 
naticcone  which  is  of  real  use  in  microscopic  illumination  where  de- 
tails are  to  be  studied.  There  is  no  doubt,  however,  that  the  results 
obtained  with  a  non-achromatic  condenser  like  the  Abbe  are  much 
more  satisfactory  than  with  no  condenser.  The  highest  results  can- 
not be  attained  with  it,  however.  ( Carpenter- Dallinger,  p.  309.) 

§  97.  Position  of  the  Condenser. — The  proper  position  of 
the  illuminator  for  high  objectives  is  one  in  which  the  beam  of  light 
traversing  it  is  brought  to  a  focus  on  the  object.  If  parallel  rays 
are  reflected  from  the  plane  mirror  to  it,  they  will  be  focused  only  a 
few  millimeters  above  the  upper  lens  of  the  condenser  ;  consequently 
the  illuminator  should  be  about  on  the  level  of  the  top  of  the  stage 
and  therefore  almost  in  contact  with  the  lower  surface  of  the  slide. 
For  some  purposes  when  it  is  desirable  to  avoid  the  loss  of  light  by 
reflection  or  refraction,  a  drop  of  water  or  homogenous  immersion 
fluid  is  put  between  the  slide  and  condenser,  forming  the  so-called 
immersion  illuminator.  This  is  necessary  only  with  objectives  of 
high  power  and  large  aperture  or  for  dark-ground  illumination. 

§  98.  Centering  the  Condenser. — The  illuminator  should  be 
centered  to  the  optic  axis  of  the  microscope,  that  is  the  optic  axis 
of  the  condenser  and  of  the  microscope  should  coincide.  Unfortun- 
ately there  is  extreme  difficulty  in  determining  when  the  Abbe 
illuminator  is  centered.  Centering  is  approximated  as  follows  : 
Put  a  pin-hole  diaphragm — that  is  a  diaphragm  with  a  small  central 
hole — over  the  end  of  the  condenser  (Fig.  58),  the  central  opening 
should  appear  to  be  in  the  middle  of  the  field  of  the  microscope.  If 


CH.  //]  LIGHTING  AND  FOCUSING  55 

it  does  not  the  condenser  should  be  moved  from  side  to  side  by 
loosening  the  centering  screws  until  it  is  in  the  center  of  the  field. 
In  case  no  pin-hole  diaphragm  accompanies  the  condenser,  one  may 
put  a  very  small  drop  of  ink,  as  from  a  pen-point,  on  the  center  of 
the  upper  lens  and  look  at  it  with  a  microscope  to  see  if  it  is  in  the 
center  of  the  field.  If  it  is  not,  the  condenser  should  be  adjusted 
until  it  is.  When  the  condenser  is  centered  as  nearly  as  possible 
remove  the  pin-hole  diaphragm  or  the  spot  of  ink.  The  microscope 
and  illuminator  axes  may  not  be  entirely  coincident  even  when  the 
center  of  the  upper  lens  appears  in  the  center  of  the  field,  as  there 
may  be  some  lateral  tilting  of  the  condenser,  but  the  above  is  the 
best  the  ordinary  worker  can  do,  and  unless  the  mechanical  arrange- 
ments of  the  illuminator  are  deficient,  it  will  be  very  nearly 
centered. 

It  is  to  be  hoped  that  the  opticians  will  devise  some  kind  of 
mounting  for  this  the  most  commonly  used  condenser  whereby  it 
may  be  centered  as  described  for  the  achromatic  condenser  instead 
of  by  the  crude  methods  described  above.  If  the  condenser  mount- 
ing regularly  possessed  centering  screws  as  in  the  microscope  of 
Watson  &  Sons  and  there  were  a  centering  diaphragm  in  the  proper 
position  so  that  its  image  could  be  projected  into  the  field  of  view, 
the  operation  would  be  very  simple.  If,  further,  the  condensers  of 
Powell  and  Lealand  were  selected  as  models  the  condensers  need  not 
be  so  bulky,  and  would  still  retain  all  their  efficiency. 

Fortunately  the  Royal  Microscopical  Society  of  L,ondon,  which 
has  done  so  much  toward  standardizing  microscopical  apparatus,  has 
proposed  a  standard  size  for  the  substage  fitting  for  the  condenser 
of  1.527  in. =38. 786 mm.  (see  §  53). 

§  99.  Mirror  and  Light  for  the  Abbe  Condenser. — It  is 
best  to  use  light  with  parallel  rays.  The  rays  of  daylight  are  prac- 
ticall}7  parallel;  it  is  best  therefore  to  employ  the  plane  mirror  for 
all  but  the  lowest  powers.  If  low  powers  are  used  the  whole  field 
might  not  be  illuminated  with  the  plane  mirror  when  the  condenser 
is  close  to  the  object  ;  furthermore,  the  image  of  the  window  frame, 
objects  outside  the  building,  as  trees,  etc.,  would  appear  with  un- 
pleasant distinctness  in  the  field  of  the  microscope.  To  overcome 
these  defects  one  can  lower  the  condenser  and  thus  light  the  object 
with  a  diverging  cone  of  light,  or  use  the  concave  mirror  and  attain 
the  same  end  when  the  condenser  is  close  to  the  object  (Fig.  54). 


56  LIGHTING  AND  FOCUSIXC  [  CH.  II 

§  100.  Artificial  Light. — If  one  uses  lamp  light,  it  is  recom- 
mended that  a  large  bull's  eye  be  placed  in  such  a  position  between 
the  light  and  the  mirror  that  parallel  rays  fall  upon  the  mirror  or  in 
some  cases  an  image  of  the  lamp  flame.  If  one  does  not  have  a 
bull's  eye  the  concave  mirror  may  be  used  to  render  the  rays  less 
divergent.  It  may  be  necessary  to  lower  the  condenser  somewhat 
in  order  to  illuminate  the  object  in  its  focus. 

ABBE   CONDENSER :    EXPERIMENTS 

§  101.  Abbe  Condenser,  Axial  and  Oblique  Light. — Use  a 
diaphragm  a  little  larger  than  the  front  lens  of  the  3  mm.  (>^in) 
objective,  have  the  illuminator  on  the  level,  or  nearly  on  the  level 
of  the  upper  surface  of  the  stage,  and  use  the  plane  mirror.  Be 
sure  that  the  diaphragm  carrier  is  in  the  notch  indicating  that  it  is 
central  in  position.  Use  the  Pleiirosigma  as  object.  Study  care- 
fully the  appearance  of  the  diatom  with  this  central  light,  then 
make  the  diaphragm  eccentric  so  as  to  light  with  oblique  light 
(§  89).  The  differences  in  appearance  will  probably  be  even  more 
striking  than  with  the  mirror  alone. 

§  102.  Lateral  Swaying  of  the  Image. — Frequently  in 
studying  an  object,  especially  with  a  high  power,  it  will  appear  to 
sway  from  side  to  side  in  focusing  up  or  down.  A  glass  stage 
micrometer  or  fly's  wing  is  an  excellent  object.  Make  the  light 
central  or  axial  and  focus  up  and  down  and  notice  that  the  lines 
simply  disappear  or  grow  dim.  Now  make  the  light  oblique,  either 
by  making  the  diaphragm  opening  eccentric  or  if  simply  a  mirror  is 
used,  by  swinging  the  mirror  sidewise.  On  focusing  up  and  down, 
the  lines  will  sway  from  side  to  side.  What  is  the  direction  of 
apparent  movement  in  focusing  down  with  reference  to  the  illumi- 
nating ray?  What  in  focusing  up?  If  one  understands  the  experi- 
ment it  may  sometimes  save  a  great  deal  of  confusion.  (See  under 
testing  the  microscope  for  swaying  with  central  light  §  130.) 

§  103.  Dark-Ground  Illumination. — When  an  object  is 
lighted  with  rays  of  a  greater  obliquity  than  can  get  into  the  front 
lens  of  the  objective,  the  field  will  appear  dark  (Fig.  57).  If  now 
the  object  is  composed  of  fine  particles,  or  is  semi-transparent,  it 
will  refract  or  reflect  the  light  which  meets  it,  in  such  a  way  that  a 


CII.  //] 


LIGHTING  AND  FOCUSING 


57 


part  of  the  very  oblique  rays  will  pass  into  the  objective,  hence  as 
light  reaches  the  objective  only  from  the  object,  all  the  surrounding 
field  will  be  dark  and  the  object  will  appear  like  a  self-luminous 
one  on  a  dark  back-ground.  This  form  of  illumination  is  most 


54  55  56  57 

FIGS.  54-57.  Sectional  vieivs  of  the  Abbe  Illuminator  of  i .20  N.A.  show- 
ing various  methods  of  illumination  (  \  101).  FIG.  54,  axial  light  with  parallel 
rays.  FIG.  55,  oblique  light.  FIG.  56,  axial  light  with  converging  beam. 
FIG.  57,  dark-ground  illumination  with  a  central  stop  diaphragm. 

Axis.  The  optic  axis  of  the  illuminator  and  of  the  microscope.  The 
illuminator  is  centered,  that  is  its  optic  axis  is  a  prolongation  of  the  optic  axis 
of  th  e  m  icroscope. 

S.  Axis.  Secondary  axis.  In  oblique  light  the  central  ray  passes  along  a 
secondary  axis  of  the  illuminator,  and  is  therefore  obliqueio  the  principal  axis. 

D.  D.  Diaphragms.  These  are  placed  in  sectional  and  in  face  views. 
The  diaphragm  is  placed  between  the  mirror  and  the  illuminator.  In  FIG.  55 
the  opening  is  eccentric  for  oblique  light,  and  in  FIG.  57  the  opening  is  a  nar- 
row ring,  the  central  part  being  stopped  out,  thus  giving  rise  to  dark-ground 
illumination  (§  /oj). 

Obj.  Obj.     The  front  of  the  objective. 

successful  with  low  powers.     It  is  well    to   make  the   illuminator 
immersion  for  this  experiment,  (see  §  116). 

(A)    With   the   Mirror. — Remove   all  the  diaphragms  so  that 


LIGHTING  AND  FOCUSING 


[  CH.  II 


very  oblique  light  may  be  used,  employ  a  stage  micrometer  in 
which  the  lines  have  been  filled  with  graphite,  use  a  16  mm. 
(-;  in.)  objective,  and  when  the  light  is  sufficiently  oblique  the 
lines  will  appear  something  like  streaks  of  s;ilver  on  a  black  back- 
ground. A  specimen  like  that  described  below  in  (B)  may  also 
be  used. 

(B)      With  the  Abbe  Condenser. — Have  the  illuminator  so  that 
the  light  is  focused  on  the  object  (see  §  97)   and  use  a  diaphragm 


FIG.  58.     Abbe  Condenser  of 1.20  FIG.  59.     Abbe  Condenser  of 1.40 

N.A.  in  section.      ^  N.A.  in  section. 

Cuts  loaned  by  Voigtldnder  &  So/in,  A.G. 

with  the  annular  opening  (Fig.  57);  employ  the  same  objective  as 
in  (A).  For  object  place  a  drop  of  10  %  solution  of  salicylic  acid 
in  95  %  alcohol  on  the  middle  of  a  slide  ;  it  will  crystallize.  The 
crystals  will  appear  brilliantly  lighted  on  a  dark  back-ground.  Put 
in  an  ordinary  diaphragm  and  make  the  light  oblique  by  making 
the  diaphragm  eccentric.  The  same  specimen  may  also  be  tried 
with  a  mirror  and  oblique  light.  In  order  to  appreciate  the  differ- 
ence between  this  dark-ground  and  ordinary  transmitted-light  illu- 
mination, use  a  central  diaphragm  and  observe  the  crystals. 

A  striking  and  instructive  experiment  may  be  made  by 
adding  a  very  small  drop  of  the  solution  to  the  dried  preparation, 
putting  it  under  the  microscope  quickly,  lighting  for  dark-ground 
illumination  and  then  watching  the  crystallization. 

'  §  1033.  Dark-Ground  Illumination  for  High  Powers.— 
There  are  two  methods  for  making  objects  appear  as  if  self  lumin- 
ous in  a  black  field  :  (i)  To  light  the  objects  by  rays  so  oblique 
that  none  of  them  will  enter  the  objective  unless  they  are  deflected 
by  some  object  in  the  field.  This  method  was  employed  above  for 
low  powers.  For  high  powers  very  wide  apertures  must  be  used 
for  the  condenser.  No  rays  below  i.oo  N.  A.  can  be  successfully 


CH.  //]  LIGHTING  AND  FOCUSING  59 

utilized.  To  accomplish  this,  Siedentopf  and  Beck  employ  a  para- 
bolic reflector  instead  of  a  condenser  of  the  usual  type.  Others 
used  condensers  specially  modified.  That  of  Reichert  is  conical  and 
silvered  on  the  conical  surface  ;  that  of  Leitz  makes  use  of  two 
internal  reflections.  By  all  these  pieces  of  apparatus  a  hollow  cone 
of  light  of  an  aperture  greater  than  i.oo  N.  A.  is  concentrated  upon 
the  field,  hence  high  powers  as  well  as  low  ones  can  be  used  pro- 
vided a  sufficiently  brilliant  source  of  light  is  employed  (sunlight, 
arc  lamp,  etc.). 

Ultramicroscopy  . — In  1903  Siedentopf  and  Zsigmondy  published 
a  method  by  which  a  further  evolution  of  dark-ground  illumination 
was  attained  according  to  the  general  principle  just  considered.  By 
their  method  the  field  is  illuminated  by  a  very  brilliant  cone  or 
wedge  of  light  from  the  side,  z.  e.,  at  right  angles  to  the  axis  of  the 
microscope.  It  is  evident  that  none  of  the  rays  can  'enter  the  micro- 
scope with  even  the  widest  apertured  objectives  unless  the  light  is  de- 
flected by  something  in  the  field.  The  brilliant  light  so  used  renders 
minute  particles  luminous  something  as  sunlight  entering  a  small 
hole  in  a  darkened  room  renders  particles  of  dust  luminous.  As 
this  method  of  lighting  rendered  particles  luminous  and  therefore 
visible  that  were  invisible  with  the  microscope  as  ordinarily  used, 
the  use  of  the  microscope  with  this  lighting  has  come  to  be  called 
Ultra  m  icroscopy . 

(2)  The  second  method  was  used  by  Toppler,  1867,  and  has 
been  revived  by  Gordon,  (J.  R.  M.  S.  1906)  and  others.  In  this 
method  the  object  is  lighted  by  a  solid  cone  of  light  from  the  con- 
denser as  usual,  but  the  aperture  of  the  condenser  must  only  fill  the 
middle  part  of  the  aperture  of  the  objective.  In  the  first  method 
the  aperture  of  the  condenser  must  be  great  and  that  of  the  objective 
moderate,  while  in  this  the  reverse  is  the  case,  and  the  objective 
should  have  a  large  aperture  and  the  condenser  a  moderate  aperture. 
The  solid  cone  of  light  used  for  illumination  has  some  of  its  rays 
deflected  by  objects  in  the  field  so  that  they  enter  the  marginal 
zones  of  the  objective.  To  secure  dark-ground  illumination  in  this 
manner  only  these  marginal  rays  are  utilized  for  the  image,  and  the 
central,  solid  cone  of  light  entering  the  objective  must  be  eliminated. 
This  is  accomplished  by  placing  a  diaphragm  or  stop  on  the  back 
lens  of  the  objective  of  just  the  right  size  to  cut  out  the  central  solid 
cone  and  allow  the  marginal  rays  to  pass  on  to  form  the  image. 


60  LIGHTING  AND  FOCUSING  [  CH.  II 

This  gives  fairly  good  results  with  all  powers.  The  same  may  also 
be  accomplished,  as  shown  by  Gordon,  1906,  by  using  a  stop  in  the 
eye-point  or  Ramsden  circle  (§  67). 

For  a  discussion  of  dark-ground  illumination  and  ultrami- 
croscopy  see  :  A.  E.  Wright,  Principles  of  Microscopy,  Ch.  XIV; 
Siedentopf,  Jour.  Roy.  Micr.  Soc.  1903,  p.  573,  1907,  p.  733  ; 
Gordon,  1906,  p.  167  ;  Beck,  1908,  p.  238  ;  Reichert,  1908,  p.  374  ; 
Leitz,  1905,  p.  502  and  Catalog  No.  42,  and  special  catalog.  Top- 
pier,  Poggendorff's  Annalen,  1867,  p.  33  ;  Beck's  Cantor  Lectures, 
1907  ;  Zeiss  special  catalog  on  Ultramicroscopy  and  dark-ground 
illumination,  1907,  gives  the  apparatus  needed,  the  methods  and 
application,  also  bibliography  ;  Cotton  et  Mouton,  Les  Ultrami- 
croscopes,  Paris,  1906. 

ARTIFICIAL   ILLUMINATION 

§  104.  For  evening  work  and  for  regions  where  daylight  is 
not  sufficiently  brilliant,  artificial  illumination  must  be  employed. 
Furthermore,  for  the  the  most  critical  investigation  of  bodies  with 
fine  markings  like  diatoms,  artificial  light  has  been  found  superior 
to  daylight. 

A  petroleum  (kerosene)  lamp  with  flat  wick  gives  a  satisfactory 
light.  It  is  recommended  that  instead  of  the  ordinary  glass  chim- 
ney one  made  of  metal  with  a  slit- opening  covered  with  an  oblong 
cover-glass  is  more  satisfactory,  as  the  source  of  light  is  more 
restricted.  Very  excellent  results  may  be  obtained,  however,  with 
the  ordinary  bed-room  lamp  furnished  with  the  usual  glass  chimney. 

The  acetylene  light  promises  to  be  excellent  for  microscopic 
observation  and  for  photo-micrography.  (See  under  photo- 
micrography.) See  also  §  ic>3a. 

Whenever  possible  the  edge  of  the  flame  is  turned  toward  the 
microscope,  the  advantage  of  this  arrangement  is  the  great  bril- 
liancy, due  to  the  greater  thickness  of  the  flame  in  this  direction. 

§  105.  Mutual  Arrangement  of  Lamp,  Bull's  Eye  and 
Microscope. — To  fulfil  the  conditions  given  above,  namely,  that 
the  object  be  illuminated  by  the  image  of  the  source  of  illumination 
the  lamp  must  be  in  such  a  position  that  the  condenser  projects  a 
sharp  image  of  the  flame  upon  the  object  (Fig.  60),  and  only  by 
trial  can  this  position  be  determined.  In  some  cases  it  is  found  ad- 


CII.  //] 


LIGHTING  AND  FOCUSING 


61 


vantageous  to  discard  the  mirror  and  allow  the  light  from  the  bull's 
eye  to  pass  directly  into  the  condenser.  In  most  cases  no  bull's  eye 
need  be  used.  The  proper  distance  of  the  lamp  from  the  mirror  and 
the  proper  elevation  of  the  condenser  give  the  required  results. 
The  position  of  lamp  and  condenser  can  be  determined  by  trial  in 
each  case. 

S   106.     Illuminating  the  Entire  Field. — With  low  objectives 
and  large  objects,  the  entire  object  might  not  be  illuminated  if  the 


Fio.  60.     i.     Lamp   with  sHt-opening   in    metal  chimney.     2.     Bull's  eye  on 
separate  stand.     3.     Screen  showing  image  of  flame, 

above  method  were  strictly  followed  ;  in  this  case  turn  the  lamp  so 
that  the  flame  is  oblique,  or  if  that  is  not  sufficient,  continue  to  turn 
the  lamp  until  the  full  width  of  the  flame  is  used.  If  necessary  the 
condenser  may  be  lowered,  and  the  concave  mirror  used.  (See 
also  §95.) 

REFRACTION    AND    COLOR    IMAGES 

I  107.  Refraction  Images  are  those  mostly  seen  in  studying  microscopic 
objects.  They  are  the  appearances  produced  by  the  refraction  of  the  light  on 
entering  and  on  leaving  an  object.  They  therefore  depend  (a)  on  the  form  of 
the  object,  (b)  on  the  relative  refractive  powers  of  object  and  mounting 
medium.  With  such  images  the  diaphragm  should  not  be  too  large  (see  ?  94). 

If  the  color  and  refractive  index  of  the  object  were  exactly  like  the  mount- 
ing medium  it  could  not  be  seen.  In  most  cases  both  refractive  index  and 
color  differ  somewhat,  there  is  then  a  combination  of  color  and  refraction 


62 


LIGHTING  AND  FOCUSING 


[  CH.  II 


images  which  is  a  great  advantage.  This  combination  is  generally  taken 
advantage  of  in  histology.  The  air  bubble  in  \  151  is  an  example  of  a  purely 
refractive  image. 

|  108.  Refraction. — laying  at  the  basis  of  microscopical  optics  is  refrac- 
tion, which  is  illustrated  by  the  above  figures.  It  means  that  light  passing 
from  one  medium  to  another  is  bent  in  its  course.  Thus  in  Fig.  61  light  pass- 
ing from  air  into  water  does  not  continue  in  a  straight  line  but  is  bent  toward 
the  normal  N-N',  the  bending  taking  place  at  the  point  of  contact  of  the  air 


N 


61.         N'  62.          N'  63.          N' 

FIGS.  61-63.  Diagrams  illustrating  refraction  in  different  media  and  at 
plane  and  curved  surfaces.  In  each  case  the  denser  medium  is  represented  by 
line  shading  and  the  perpendicular  or  normal  to  the  refracting  surface  is  repre- 
sented by  the  doited  line  N-N' ,  the  refracted  ray  by  the  bent  line  A  C. 

and  water ;  that  is,  the  ray  of  light  A  B  entering  the  water  at  B  is  bent  out  of 
its  course,  extending  to  C  instead  of  Cx. 

Conversely,  if  the  ray  of  light  is  passing  from  water  into  air,  on  reaching 
the  air  it  is  bant  from  the  normal,  the  ray  C  B  passing  to  A  and  not  in  a 
straight  line  to  Cx/.  By  comparing  Figs.  62-63  in  which  the  denser  medium  is 
crown  glass  instead  of  water,  the  bending  of  the  rays  is  seen  to  be  greater  as 
crown  glass  is  denser  than  water. 

It  has  been  found  by  physicists  that  there  is  a  constant  relation  between 
the  angle  taken  by  the  ray  in  the  rarer  medium  and  that  taken  by  the  ray  in 
the  denser  medium.  The  relationship  is  expressed  thus:  Sine  of  the  angle  of 
incidence  divided  by  the  sine  of  the  angle  of  refraction  equals  the  index of '  re- 

Sin  AB N 

fraction.  In  the  figures,  -^ — T^DITV  =  index   of  refraction, 
oin  Lor* 


Worked  out  com- 


pletely in  Fig.  61,  A  B  N=&°,  CB  A"=28°  54'  and 


Sin  40° 


0.6427 
0.48327 


Sin  28°  54'" 

1.33,  i.  <?.,  the  index  of  refraction  from  air  to  water  is  1.33.  (See  \  39.)  In 
Figs.  62-63,  illustrating  refraction  in  crown  glass,  the  angles  being  given, 
the  problem  is  easily  solved  as  just  illustrated.  (For  table  of  natural  sines  see 
third  page  of  cover  ;  for  interpolation,  \  38. ) 

§  109.     Absolute  Index  of  Refraction. — This  is  the  index  of  refraction  ob- 


C //.//]  LIGHTING  AND  FOCUSING  63 

tained  when  the  incident  ray  passes  from  a  vacuum  into  a  given  medium.  As 
the  index  of  the  vacuum  is  taken  as  unity,  the  absolute  index  of  any  substance 
is  always  greater  than  unity.  For  many  purposes,  as  for  the  object  of  this 
book,  air  is  treated  as  if  it  were  a  vacuum,  and  its  index  ie  called  unity,  but  in 
reality  the  index  of  refraction  of  air  is  about  3  ten-thousandths  greater  than 
unity.  Whenever  the  refractive  index  of  a  substance  is  given,  the  absolute 
index  is  meant  unless  otherwise  stated.  For  example,  when  the  index  of 
refraction  of  water  is  said  to  be  1.33,  and  of  crown  glass  1.52,  etc.,  these  figures 
represent  the  absolute  index,  and  the  incident  ray  is  supposed  to  be  in  a 
vacuum. 

\  1 10.  Relative  Index  of  Refraction. — This  is  the  index  of  refraction  be- 
tween two  contiguous  media,  as  for  example  between  glass  and  diamond, 
water  and  glass,  etc.  It  is  obtained  by  dividing  the  absolute  index  of  refrac- 
tion of  the  substance  containing  the  refracted  ray,  by  the  absolute  index  of 
the  substance  transmitting  the  incident  ray.  For  example,  the  relative  index 
from  water  to  glass  is  1.52  divided  by  1.33.  If  the  light  passed  from  glass  to 
water  it  would  be,  1.33  divided  by  1.52. 

By  a  study  of  the  figures  showing  refraction,  it  will  be  seen  that  the 
greater  the  refraction  the  less  the  angle  and  consequently  the  less  the  sine  of 
the  angle,  and  as  the  refraction  between  two  media  is  the  ratio  of  the  sines  of 

the  angles  of  incidence  and  refraction  (  J ,  it  will  be  seen  that  whenever 

\sm  r/ 

the  sine  of  the  angle  of  refraction  is  increased  by  being  in  a  less  refractive 
medium,  the  index  of  refraction  will  show  a  corresponding  decrease  and  vice 
versa.  That  is  the  ratio  of  the  sines  of  the  angles  of  incidence  and  refraction  of 
any  two  contiguous  substances  is  inversely  as  the  refractive  indices  of  those  sub- 
stances. The  formula  is : 

/  Sine  of  angle  of  incident  ray  \       /  Inedx  of  refraction  of  refracting  medium  \ 
\Sineofangleof  refracted  ray  /  ~~  \Index  of  refraction  of  incident  medium  / 

,  /sin  i\       / index  r  \ 

Abbreviated  (  -. I  =  I   .-       — -   I  .      By  means  of  this  general  formula  one 

\sinr/       \  index*    / 

can  solve  any  problem  in  refraction  whenever  three  factors  of  the  problem  are 
known.     The  universality  of  the  law  may  be  illustrated  by  the  following  cases  : 
(A)     Light  incident  in  a  vacuum  or  in  air,  and  entering   some   denser 
medium,  as  water,  glass,  diamond,  etc. 

/     Sine  of  angle  made  by  the  ray  in  air     \_(  Index  of  ref.  of  denser  med  \ 
V  Sine  of  anglelnadeTbyTay  in^lenser  med.  /      \     Index  of  ref.  of  air  (i )     / 

If  the  dense  substance  were  glass  (  S      -  )  =  (  ^^  )  •     If  the  two  media  were 

\sm  rj      \     i      / 

weter   and  glass,  the  incident  light  being  in  water  the  formula  would   be  ; 

(sl^\  —  (  I-$2  \    .      if  the  incident  ray  were  glass  and  the  refracted  ray 
\sin  rj      \  1.33  / 

in  water-    /sln  *\_/.I:33_\  _     An(j  similarly  for  any  two  media;    and  as 

\sin  r/       \  I.52  / 

stated  above  if  any  three  of  the  factors  are  given  the  fourth  may  be  readily 
found. 


64  LIGHTING  AND  FOCUSING  [  CH.  II 

?  in.  Critical  Angle  and  Total  Reflection. — In  order  to  understand  the 
Wollaston  camera  lucida  (Ch.  IV)  and  other  totally  reflecting  apparatus,  it  is 
necessary  briefly  to  consider  the  critical  angle. 

The  critical  angle  is  the  greatest  angle  that  a  ray  of  light  in  the  denser  of 
two  contiguous  media  can  make  with  the  normal  and  still  emerge  into  the 
less  refractive  medium.  On  emerging  it  will  form  an  angle  of  90°  with  the 
normal,  and  if  the  substances  are  liquids,  the  refracted  ray  will  be  parallel 
with  the  surface  of  the  denser  medium. 

Total  Reflection. — In  case  the  incident  ray  in  the  denser  medium  is  at  an 
angle  with  the  normal  greater  than  the  critical  angle,  it  will  be  totally  reflected 
at  the  surface  of  the  denser  medium,  that  surface  acting  as  a  perfect  mirror. 
By  consulting  the  figures  it  will  be  seen  that  there  is  no  such  thing  as  a 
critical  angle  and  total  reflection  in  the  rarer  of  two  contiguous  media. 

To  find  the  critical  angle  in  the  denser  of  two  contiguous  media :  — 

Make  the  angle  of  refraction  (i.   e.,  the  angle  in  the   rarer  of  the  two 

/sin  i\       /  index  r  \ 

media)  90°  and  solve  the  general  equation  :(          -)  =  (-;  r).    Ivet  the 

\sm  r/      \  index  i  / 

two  substances  be  water  and  air,  then  the  sine  of  r( 90°) is  i,  and  the  index  of  air 
is  i,  that  of  water  1.33,  whence/          -  j=f  -      -  j    or  sin  z— 751  +  .      This   is 

the  sine  of  48°+,  and  whenever  the  ray  in  the  water  is  at  an  angle  of  more 

than  48°  it  will   not  emerge  into  the  air,  but  be  totally  reflected  back  into 

the  water. 

The  case  of  a  ray  passing  from  crown  glass  into  the  water  : 
/  sin  i  X^/ index  water  (1.33)  \  Qr /sin_«'\  _/_i.33\ 

\  sin  r  (sin  90°— -i)/ ~~\  index  glass  (1.52)7       \      i     /  '    \  1.52 /' 

whence  sin  i— .875  sine  of  critical  angle  in  glass  covered  with  water.     The 

corresponding  angle  is  approximately  61°. 

\  112.  Color  Images.  These  are  images  of  objects  which  are  strongly 
colored  and  lighted  with  so  wide  an  aperture  that  the  refraction  images  are 
drowned  in  the  light.  Such  images  are  obtained  by  removing  the  diaphragm 
or  by  using  a  larger  opening.  This  method  of  illumination  is  especially 
applicable  to  the  study  of  deeply  stained  bacteria.  (See  below  §  119.) 

ADJUSTABLE,    WATER  AND    HOMOGENEOUS   OBJECTIVES  : 
EXPERIMENTS 

.  \  113.  Adjustment  for  Objectives.  As  stated  above  (%  27),  the  aberration 
produced  by  the  cover-glass  (Fig.  64),  is  compensated  for  by  giving  the  com- 
binations in  the  objective  a  different  relative  position  than  they  would  have  if 
the  objective  were  to  be  used  on  uncovered  objects.  Although  this  relative 
position  cannot  be  changed  in  unadjustable  objectives,  one  can  secure  the 
best  results  of  which  the  objective  is  capable  by  selecting  covers  of  the  thick- 
ness for  which  the  objective  was  corrected.  (See  table  §33.)  Adjustment 
may  be  made  also  by  increasing  the  tube-length  for  covers  thinner  than  the 


CH.  11} 


LIGHTING  AND  FOCUS  l.\(, 


standard   and    by   shortening   the    tube-length    for   covers   thicker  than    the 
standard  (Fig.  65). 

In  learning  to  adjust  objectives,  it  is  best  for  the  student  to  choose  some 
object  whose  structure  is  well  agreed  upon,  and  then  to  practice  lighting  it, 
shading  the  stage  and  adjusting  the  objective,  until  the  proper  appearance  is 
obtained.  The  adjustment  is  made  by  turning  a  ring  or  collar  which  acts  on  a 
screw  and  increases  or  diminishes  the  distance  between  the  systems  of  lenses, 
usually  the  front  and  the  back  systems  (Fig.  45). 

FIG.  64.  Effect  of  the  cover- 
g/ass  on  the  rays  from  the  object  to 
the  objective  (Ross). 

Axis.  The  projection  of  the 
optic  axis  of  the  microscope. 

F.  Focal  or  axial  point  of  the 
objective. 

F'  and  F" .  Points  on  the  a.ris 
where  rays  2  and  j  appear  to  orig- 
inate if  traced  backward  after 
emerging  from  the  upper  side  of 
the  cover-glass. 

§  114.  Directions  for  Adjustment. — (A)  The  thinner  the 
cover-glass,  the  further  must  the  systems  be  separated,  i.  e.,  the  ad- 
justing collar  is  turned  nearer  the  zero  or  the  mark  "  uncovered," 
and  conversely;  (B)  the  thicker  the  cover-glass  the  closer  together 
are  the  systems  brought  by  turning  the  adjusting  collar  from  the 
zero  mark.  This  also  increases  the  magnification  of  the  objective 
(Ch.  IV). 

The  following  specific  directions  for  making  the  cover- glass  ad- 
justment are  given  by  Mr.  Wenham  (Carpenter,  yth  Ed.,  p.  166). 
"  Select  any  dark  speck  or  opaque  portion  of  the  object,  and  bring 
the  outline  into  perfect  focus;  then  lay  the  finger  on  the  milled-head 
of  the  fine  motion,  and  move  it  briskly  backwards  and  forwards  in 
both  directions  from  the  first  position.  Observe  the  expansion  of 
the  dark  outline  of  the  object,  both  when  within  and  when  without 
the  focus.  If  the  greater  expansion  or  coma  is  when  the  object  is 
without  the  focus,  or  farthest  from  the  objective  [z.  e.,  in  focusing 
up] ,  the  lenses  must  be  placed  further  asunder,  or  toward  the  mark 
uncovered  [the  adjusting  collar  is  turned  toward  the  zero  mark  as 
the  cover-glass  is  too  thin  for  the  present  adjustment] .  If  the 
greater  expansion  is  when  the  object  is  within  the  focus,  or  nearest 
the  objetive  [i.e.,  in  focusing  down],  the  lenses  must  be  brought 


66  LK;HTINC;  AND  FOCTSING  \_cti.  n 

closer  together,  or  toward  the  mark  covered  [?'.  e. ,  the  adjusting 
collar  should  be  turned  away  from  the  zero  mark,  the  cover-glass 
being  too  thick  for  the  present  adjustment]."  In  most  objectives  the 
collar  is  graduated  arbitrarily,  the  zero  ((9)  mark  representing  the  posi- 
tion/or uncovered  objects.  Other  objectives  have  the  collar  graduated  to 
correspond  to  the  various  thickness  of  cover-glasses  for  which  the  ob- 
jective may  be  adjusted.  This  seems  to  be  an  admirable  plan;  then  if 
one  knows  the  thickeness  of  the  cover-glass  on  the  preparation  (C/i. 
VIII )  the  adjusting  collar  may  be  set  at  a  corresponding  mark,  and 
one  will  feel  confident  that  the  adjustment  will  be  approximately  cor- 
rect. It  is  then  only  necessary  for  the  observer  to  make  the  slight  ad- 
justment to  compensate  for  the  mounting  medium  or  any  variation 
from  the  standard  length  of  the  tube  of  the  microscope.  In  adjusting 
for  variations  of  the  length  of  the  tube  from  the  standard  it  should 
be  remember  that  :  (A)  If  the  tube  of  the  microscope  is  longer 
than  the  standard  for  which  the  objective  was  corrected,  the  effect 
is  approximately  the  same  as  thickening  the  cover-glass,  and  there- 
fore the  systems  of  the  objective  must  be  brought  closer  together,  i. 
e.,  the  adjusting  collar  must  be  turned  away  from  the  zero  mark. 
(B)  If  the  tube  is  shorter  than  the  standard  for  which  the  objective 
is  corrected,  the  effect  is  approximately  the  same  as  diminishing  the 
thickness  of  the  cover-glass,  and  the  systems  must  therefore  be 
separated  (Fig.  45). 

In  using  the  tube-length  for  cover  correction  Shorten  the  tube 
for  too  thick  covers  and  Lengthen  the  tube  for  too  thin  covers. 

Furthermore,  whatever  the  interpretation  by  different  opticians 
of  what  should  be  included  in  "  tube-length,"  and  the  exact  length 
in  millimeters,  its  importance  is  very  great;  for  each  objective  gives 
the  most  perfect  image  of  which  it  is  capable  with  the  "tube- 
length  "  for  which  it  is  corrected,  and  the  more  perfect  the  objective 
the  greater  the  ill-effects  on  the  image  of  varying  the  "tube-length" 
from  the  standard.  The  plan  of  designating  exactly  what  is  meant 
by  "tube-length,"  and  engraving  on  each  objective  the  "tube- 
length  for  which  it  is  corrected,  is  to  be  commended,  for  it  is  mani- 
festly difficult  for  each  worker  with  the  microscope  to  find  out  for 
himself  for  what  "tube-length"  each  of  his  objectives  was  cor- 
rected. (SeeCh.  X.) 

§  115.  Water  Immersion  Objectives. — Put  a  water  immer- 
sion objective  in  position  (§  54)  and  the  fly's  wing  for  object  under 


C//.  //] 


AND  FOCUSING 


the  microscope.  Place  a  drop  of  distilled  water  on  the  cover-glass, 
and  with  the  coarse  adjustment  lower  the  tube  till  the  objective 
dips  into  the  water,  then  light  the  field  well  and  turn  the  fine  ad- 
justment one  way  and  another  till  the  image  is  clear.  Water  im- 
mersions are  exceedingly  convenient  in  studying  the  circulation  of 
the  blood,  and  for  many  other  purposes  where  aqueous  liquids  are 


Objective 


Object-b 
Object-a 

FIG.  65.  Figure  to  show  thai  in  lengthening  the  tube\of  the  microscope  the 
object  must  be  brought  nearer  the  principal  focus  or  center  of  the  lens.  It  will 
be  seen  by  consulting  the  figure  that  in  shortening  the  tube  of  the  microscope  the 
object  must  be  removed  farther  from  the  center  of  the  lens.  By  consulting  the 
figure  showing  the  effect  of  the  cover-glass  (Fig.  64)  it  will  be  seen  that  the 
effect  of  the  cover-glass  is  to  bring  the  object  nearer  the  objective,  and  the  thicker 
the  cover  the  nearer  is  the  object  brought  to  the  objective.  As  shortening  the 
tube  serves  to  remove  the  object,  it  neutralizes  the  effect  of  the  thick  cover,  and  if 
the  cover  is  so  thin  that  it  does  not  elevate  the  object  enough  for  the  corrections 
of  the  objective,  then  an  increase  in  the  tube-length  will  correct  the  defect. 


68  LIGHTING  AND  FOCUSING  [  CH.  If 

liable  to  get  on  the  cover-glass.     If  the  objective  is  adjustable,  fol- 
low the  directions  given  in  §  114. 

When  one  is  through  using  a  water  immersion  objective, 
remove  it  from  the  microscope  and  with  some  lens  paper  wipe  all 
the  water  from  the  front  lens.  Unless  this  is  done  dust  collects  and 
sooner  or  later  the  front  lens  will  be  clouded.  It  is  better  to  use 
distilled  water  to  avoid  the  gritty  substances  that  are  liable  to  be 
present  in  natural  waters,  as  these  gritty  particles  might  scratch  the 
front  lens. 

HOMOGENEOUS    IMMERSION    OBJECTIVES  :    EXPERIMENTS 

§  116.  As  stated  above,  these  are  objectives  in  which  a  liquid 
of  the  same  refractive  index  as  the  front  lens  of  the  objective  is 
placed  between  the  front  lens  and  the  cover-glass. 

§  117.  Tester  for  Homogeneous  Liquid. — In  order  that 
full  advantage  be  derived  from  the  homogeneous  immersion  prin- 
ciple, the  liquid  employed  must  be  truly  homogeneous.  To  be  sure 
that  such  is  the  case,  one  may  use  a  tester  like  that  constructed  by 
the  Gundlach  Optical  Co.,  then  if  the  liquid  is  too  dense  it  may  be 
properly  diluted  and  vice  versa.  For  the  cedar  oil  immersion  liquid, 
the  density  may  be  diminished  by  the  addition  of  pure  cedar  wood 
oil.  The  density  may  be  increased  by  allowing  it  to  thicken  by 
evaporation.  (See  H.  L,.  Smith,  Proc.  Amer.  Soc.  Micr.,  1885,  p. 
83,  and  Ch.  X.) 

§118.  Refraction  Images. — Put  a  2  mm.  (TVin.)  homo- 
geneous immersion  objective  in  position,  employ  an  illuminator. 
Use  some  histological  specimen  like  a  muscular  fiber  as  object, 
make  the  diaphragm  opening  about  9  mm.  in  diameter,  add  a  drop 
of  the  homogeneous  immersion  liquid  and  focus  as  directed  in  §  83. 
The  object  will  be  clearly  seen  in  all  details  by  the  unequal  refrac- 
tion of  the  light  traversing  it.  The  difference  in  color  between  it 
and  the  surrounding  medium  will  also  increase  the  sharpness  of  the 
outline.  If  an  air  bubble  preparation  (§  88)  were  used,  one  would 
get  pure,  refraction  images. 

§  119.  Color  Images. — Use  some  stained  bacteria  as  Bacillus 
tuberculosis  for  object.  Put  a  drop  of  the  immersion  liquid  on  the 
cover-glass  or  the  front  lens  of  the  homogeneous  objective.  Re- 


CH.  //] 


/./(; //TING  AND  FOCUSING 


69 


move  the  diaphragms  from  the  illuminator  or  in  case  the  iris  dia- 
phragm is  used,  open  to  its  greatest  extent.  Focus  the  objective 
down  so  that  the  immersion  fluid  is  in  contact  with  both  the  front 
lens  and  the  cover-glass,  then  with  the  fine  adjustment  get  the 
bacteria  in  focus.  The}'  will  stand  out  as  clearly  defined  colored  ob- 
jects on  a  bright  field. 


FIG.  66.  Screen  for  shading  the  microscope  and 
the  face  of  the  observer.  This  is  very  readily  con- 
structed as  shown  in  the  figure  by  supporting  a  wire 
in  a  disc  of  lead,  iron,  or  heavy  wood.  The  screen  is 
then  completed  by  hanging  over  the  bent  wire,  black 
cloth  or  paper  jo  x  40  cm.  The  lower  edge  of  the 
screen  should  be  a  little  below  the  stage  of  the  micro- 
scope and  the  tipper  edge  high  enough  to  screen  the 
eyes  of  the  observer. 


jo    cm 


§  120.  Shading  the  Object. — To  get  the  clearest  image  of  an 
object  no  light  should  reach  the  eye  except  from  the  object.  A 
handkerchief  or  a  dark  cloth  wound  around  the  objective  will  serve 
the  purpose.  Often  the  proper  effect  may  be  obtained  by  simply 
shading  the  top  of  the  stage  with  the  hand  or  with  a  piece  of  bristol 
board.  Unless  one  has  a  very  favorable  light  the  shading  of  the 
object  is  of  the  greatest  advantage,  especially  with  homogeneous 
immersion  objectives.  The  screen  (Fig.  66)  is  the  most  satisfactory 
means  for  this  purpose,  as  the  entire  microscope  above  the  illuminat- 
ing apparatus  is  shaded. 

§  121.  Cleaning  Homogeneous  Objectives. — After  one  is 
through  with  a  homogeneous  objective,  it  should  be  carefully  cleaned 
as  follows:  Wipe  off  the  homogeneous  liquid  with  a  piece  of  the  lens 
paper  (§  125),  then  if  the  fluid  is  cedar  oil,  wet  one  corner  of  a  fresh 
piece  in  xylene  or  chloroform  and  wipe  the  front  lens  with  it.  Im- 
mediately afterward  wipe  with  a  dry  part  of  the  paper.  The  cover- 
glass  of  the  preparation  can  be  cleaned  in  the  same  way.  If  the 
homogeneous  liquid  is  a  glycerin  mixture  proceed  as  above,  but  use 
water  to  remove  the  last  traces  of  glycerin. 


70  LIGHTING  AND  FOCUSING  [  CH.    II 

CARE    OF   THE    MICROSCOPE 

§  122.  The  microscope  should  be  handled  carefully  and  kept 
perfectly  clean.  The  oculars  and  objectives  should  never  be  allowed 
to  fall. 

When  not  in  use  keep  it  in  a  place  as  free  as  possible  from  dust. 

All  parts  of  the  microscope  should  be  kept  free  from  liquids, 
especially  from  acids,  alkalies,  alcohol,  xylene,  turpentine  and 
chloroform. 

§  123.  Care  of  the  Mechanical  Parts. — To  clean  the  mechan- 
ical parts  put  a  small  quantity  of  some  fine  oil  (olive  oil  or  liquid 
vaselin  and  gasoline  or  xylene,  equal  parts),  on  a  piece  of  chamois 
leather  or  on  the  lens  paper,  and  rub  the  parts  well,  then  with  a 
clean  dry  piece  of  the  chamois  or  paper  wipe  off  most  of  the  oil.  If 
the  mechanical  parts  are  kept  clean  in  this  way  a  lubricator  is  rarely 
needed.  When  opposed  brass  surfaces  "cut,"  i.  <?.,  when  from  the 
introduction  of  some  gritty  material,  minute  grooves  are  worn  in  the 
opposing  surfaces,  giving  a  harsh  movement,  the  opposing  parts 
should  be  separated,  carefully  cleaned  as  described  above  and  any 
ridges  or  prominences  scraped  down  with  a  knife.  Where  the  ten- 
dency to  "  cut  "  is  marked,  a  very  slight  application  of  equal  parts 
of  beeswax  and  tallow,  well  melted  together,  serves  a  good  purpose. 

In  cleaning  lacquered  parts,  xylene  alone  answers  well,  but  it 
should  be  quickly  wiped  off  with  a  clean  piece  of  the  lens  paper. 
Do  not  use  alcohol  as  it  dissolves  the  lacquer. 

§  124.  Care  of  the  Optical  Parts. — These  must  be  kept 
scrupulously  clean  in  order  that  the  best  results  may  be  obtained. 

Glass  surfaces  should  never  be  touched  with  the  fingers,  for 
that  will  soil  them. 

The  glass  of  which  the  lenses  are  made  is  quite  soft,  consequent- 
ly it  is  necessary  that  only  soft,  clean  cloth  or  paper  be  used  in 
in  wiping  them. 

Whenever  an  objective  is  left  in  position  on  a  microscope,  or 
when  several  are  attached  by  means  of  a  revolving  nose-piece,  an 
ocular  should  be  left  in  the  upper  end  of  the  tube  to  prevent  dust 
from  falling  down  upon  the  back  lens  of  the  objective. 

§  125.  Lens  Paper. — The  so-called  Japanese  filter  paper, 
which  from  its  use  with  the  microscope,  I  have  designated  lens  paper, 


CH.  //]  LIGHTING;  AND  FOCUSING  71 

has  been  used  in  the  author's  laboratory  since  1885  for  cleaning  the 
lenses  of  oculars  and  objectives,  and  especially  for  removing  the 
fluid  used  with  immersion  objectives.  Whenever  a  piece  is  used 
once  it  is  thrown  away.  It  has  proved  more  satisfactory  than  cloth 
or  chamois,  because  dust  or  sand  is  not  present;  and  from  its  bib- 
ulous character  it  is  very  efficient  in  removing  liquid  or  semi-liquid 
substances. 

S  126.  Removal  of  Dust. — Dust  may  be  removed  with  a 
camel's  hair  brush,  or  by  wiping  with  the  lens  paper. 

Cloudiness  may  be  removed  from  the  glass  surfaces  by  breathing 
on  them,  then  wiping  quickly  with  a  soft  cloth  or  the  lens  paper. 

Cloudiness  on  the  inner  surfaces  of  the  ocular  lenses  may  be 
removed  by  unscrewing  them  and  wiping  as  directed  above.  A 
high  objective  should  never  be  taken  apart  by  an  inexperienced 
person. 

If  the  cloudiness  cannot  be  removed  as  directed  above,  moisten 
one  corner  of  the  cloth  or  paper  with  95  per  cent  alcohol,  wipe  the 
glass  first  with  this,  then  with  the  dry  cloth  or  the  paper. 

Water  may  be  removed  with  soft  cloth  or  the  paper. 

Glycerin  may  be  re  moved- with  cloth  or  paper  saturated  with  dis- 
tilled water;  remove  the  water  as  above. 

Blood  or  other  albuminous  material  may  be  removed  while  tresh 
with  a  moist  cloth  or  paper,  the  same  as  glycerin.  If  the  material 
has  dried  on  the  glass,  it  may  be  removed  more  readily  by  adding  a 
small  quantity  of  ammonia  to  the  water  in  which  the  cloth  is  moist- 
ened, (water  100  cc.,  ammonia  i  cc). 

Canada  Balsam,  damar,  paraffin,  or  any  oily  substance  may  be 
removed  with  a  cloth  or  paper  wet  with  chloroform,  gasoline  or 
xylene.  The  application  of  these  liquids  and  their  removal  with  a 
soft  dry  cloth  or  paper  should  be  as  rapid  as  possible,  so  that  none 
of  the  liquid  will  have  time  to  soften  the  setting  of  the  lenses. 

Shellac  Cememt  may  be  removed  by  the  paper  or  a  cloth  moist- 
ened in  95  per  cent,  alcohol. 

Brunswick  Black,  Gold  Size,  and  all  other  substances  soluble  in 
chloroform,  etc.,  may  be  removed  as  directed  for  balsam  and  damar. 

In  general,  use  a  solvent  of  the' substance  on  the  glass  and  wipe 
it  off  quickly  with  a  fresh  piece  of  the  lens  paper. 


LIGHTING  AND  FOCUSING 


\_Cff.  II 


It  frequently  happens  that  the  upper  surface  of  the  back  com- 
bination of  the  objective  becomes  dust}7.  This  may  be  removed  in 
part  by  a  brush,  but  more  satisfactorily  by  using  a  piece  of  the  soft 
paper  loosely  twisted.  When  most  of  the  dust  is  removed  some  of 
the  paper  may  be  put  over  the  end  of  a  pine  stick  (like  a  match 
stick)  and  the  glass  surfaces  carefully  wiped. 


CARE    OF   THE  EYES 

§  127.  Keep  both  eyes  open,  using  the  eye-screen  if  necessary 
'(Fig.  67);  and  divide  the  labor  between  the  two  eyes,  i.  e. 
use  one  eye  for  observing  the  image  awhile  and  then  the  other.  In 
the  beginning  it  is  not  advisable  to  look  into  the  microscope  con- 
tinuously for  more  than  half  an  hour  at  a  time.  One  never  should 
work  with  the  microscope  after  the  eyes  feel  fatigued.  After  one 


FIG.  67.  Adjusting  Eye-Shade.  This  is  prepared  by  covering  a  card 
about  6x12  centimeters  with  black  velveteen.  A  copper  wire  about  j  mm. 
(l/&  in.}  and  of  the  right  length  is  curved  as  shown  in  the  figure.  Its 
ends  are  rounded,  and  finally  it  is  put  tmder  the  cloth  and  sewed  carefully  all 
around.  The  card  and  cloth  are  then  cut  as  shown.  The  flexible  wire  makes 
it  possible  to  put  the  screen  on  the  tube  at  any  level. 

becomes  accustomed  to  microscopic  observation  he  can  work  for 
several  hours  with  the  microscope  without  fatiguing  the  eyes.  This 
is  due  to  the  fact  that  the  eyes  become  inured  to  labor  like  the  other 
organs  of  the  body  by  judicious  exercise.  It  is  also  due  to  the  fact 
that  but  very  slight  accomodation  is  required  of  the  eyes,  the  eyes 
remaining  nearly  in  a  condition  of  rest  as  for  distant  objects.  The 


CH.  //] 


LIGHTING  AND  FOCUSING 


73 


fatigue  incident  upon  using  the  microscope  at  first  is  due  partly  at 
least  to  the  constant  effort  on  the  part  of  the  observer  to  remedy 
the  defects  of  focusing  the  microscope  by  accommodation  of  the 
eyes.  This  should  be  avoided  and  the  fine  adjustment  of  the  micro- 
scope used  instead  of  the  muscles  of  accommodation.  With  a  micro- 
scope of  the  best  quality,  and  suitable  light — that  is  light  which  is 
steady  and  not  so  bright  as  to  dazzle  the  eyes  nor  so  dim  as  to  strain 
them  in  determining  details— microscopic  work  should  improve 
rather  than  injure  the  sight. 

FIG.    68.      Laboratory    Table 
with  adjustable  stool. 

§  128.     Position  and  Char- 
acter of  the  Work  Table.— 

The  work-table  should  b  e 
very  firm  and  large  (60  x  120 
cm.;  24  x  48  in.),  so  that  the 
necessary  apparatus  and  ma- 
terial for  work  may  not  be 
too  crowded.  The  table 
should  also  be  of  the  right 
height  to  make  work  by  it 
comfortable.  An  adjustable 
stool,  something  like  a  piano 
stool  is  convenient,  then  one 
may  vary  the  height  corres- 
ponding to  the  necessities  of  special  cases.  It  is  a  great  advantage 
to  sit  facing  the  window  if  daylight  is  used,  then  the  hands  do  not 
constantly  interfere  with  the  illumination.  To  avoid  the  discomfort 
of  facing  the  light  a  screen  like  that  shown  here  and  in  Fig.  66  is 
very  useful  (see  also  under  lighting,  §  71). 

TESTING  THE    MICROSCOPE 

§  129.  Testing  the  Microscope. — To  be  of  real  value  this  must  be  accom- 
plished by  a  person  with  both  theoretical  and  practical  knowledge,  and  also 
with  an  unprejudiced  mind.  Such  a  person  is  not  common,  and  when  found 
does  not  show  over  anxiety  to  pass  judgement.  Those  most  ready  to  offer  ad- 
vice should  as  a  rule  be  avoided,  for  in  most  cases  they  simply  "have  an  ax  to 
grind,"  and  are  sure  to  commend  only  those  instruments  that  conform  to  the 
"  fad"  of  the  day.  From  the  writer's  experience  it  seems  safe  to  say  that  the 


LABORATORY       TABLL 


74  LIGHTING  AND  FOCUSING  [  CH.  II 

inexperienced  can  do  no  better  than  to  state  clearly  what  he  wishes  to  do  with 
a  microscope  and  then  trust  to  the  judgement  of  one  of  the  optical  companies. 
The  makers  of  microscopes  and  objectives  guard  with  jealous  care  the  excel- 
lence of  both  the  mechanical  and  optical  part  of  their  work,  and  send  out  only 
instruments  that  have  been  carefully  tested  and  found  to  conform  to  the  stand- 
ard. This  would  be  done  as  a  matter  of  business  prudence  on  their  part,  but 
it  is  believed  by  the  writer  that  microscope  makers  are  artists  first  and  take  an 
artist's  pride  in  their  work;  they  therefore  have  a  stimulus  to  excellence 
greater  than  business  prudence  alone  could  give. 

\  130.  Mechanical  Parts. — All  of  the  parts  should  be  firm,  and  not  too 
easily  shaken.  Bearings  should  work  smoothly.  The  mirror  should  remain 
in  any  position  in  which  it  is  placed. 

Focusing  Adjustments. — The  coarse  or  rapid  adjustment  should  be  by  rack 
and  pinion,  and  work  so  smoothly  that  even  the  highest  power  can  be  easily 
focused  with  it.  In  no  case  should  it  work  so  easily  that  the  body  of  the 
micoscope  is  liable  to  run  down  and  plunge  the  objective  into  the  object.  If 
any  of  the  above  defects  appear  in  a  microscope  that  has  been  used  for  some 
time,  a  person  with  moderate  mechanical  instinct  will  be  able  to  tighten  the 
proper  screw,  etc. 

The  Fine  Adjustment  is  more  difficult  to  deal  with.  From  the  nature  of 
its  purpose  unless  it  is  approximately  perfect,  it  would  be  better  off  the  micro- 
scope entirely.  It  has  been  much  improved  recently. 

It  should  work  smoothly  and  be  so  balanced  t,hat  one  cannot  tell  by  the 
feeling  when  using  it  whether  the  screw  is  going  up  or  down.  Then  there 
should  be  absolutely  no  motion  except  in  the  direction  of  the  optic  axis,  other- 
wise the  image  will  appear  to  sway  even  with  central  light.  Compare  the  ap- 
pearance when  using  the  coarse  and  when  using  the  fine  adjustment.  There 
should  be  no  swaying  of  the  image  with  either  if  the  light  is  central  (£  88). 

|  131.  Testing  the  Optical  Parts. — As  stated  in  the  beginning,  this  can  be 
done  satisfactorily  only  by  an  expert  judge.  It  would  be  of  very  great  advant- 
age to  the  student  if  he  could  have  the  help  of  such  a  person.  In  no  case  is  a 
microscope  to  be  condemned  by  an  inexperienced  person.  If  the  beginner  will 
bear  in  mind  that  his  failures  are  due  mostly  to  his  own  lack  of  knowledge 
and  lack  of  skill;  and  will  truly  endeavor  to  learn  and  apply  the  principles 
laid  down  in  this  and  in  the  standard  works  referred  to,  he  will  learn  after  a 
while  to  estimate  at  their  true  value  all  the  pieces  of  his  microscope.  (See 
Ch.  X). 


LABORATORY  AND  HIGH-SCHOOL  COMPOUND 
MICROSCOPES 


\  132.     Optical  Parts. — A  great  deal   of  beginning   work  with  the  micro 
scope  in  biological  laboratories  is  done  with  simple  and  inexpensive  apparatus. 


r//.  //]  LIGHTING  AND  FOfUSING  75 

Indeed  if  one  contemplates  the  large  classes  in  the  high  schools,  the  universi- 
ties and  medical  schools,  it  can  be  readily  understood  that  microscopes  costing 
from  $25  to  50  each  and  magnifying  from  25  to  500  diameters,  are  all  that  can  be 
expected.  But  for  the  purpose  of  modern  histological  investigation  and  of  ad- 
vanced microscopical  work  in  general,  a  microscope  should  have  something 
like  the  following  character:  Its  optical  outfit  should  comprise,  (a)  dry  objec- 
tives of  50  mm.  (2  in.),  16-18  mm.  ( -' ;  in.)  and  3  mm.  (  's  in  )  equivalent  focus. 
There  should  be  present  also  a  2  mm.  (  ,'._.  in.  )  or  1.5  mm.  (,',.  in.  )  homogeneous 
immersion  objective.  Of  oculars  there  should  be  several  of  different  power. 
A  centering  substage  condenser,  and  an  Abbe  camera  lucida  are  also  neces- 
sities, and  a  micro-spectroscope  and  a  micro-polarizer  are  very  desirable. 

Even  in  case  all  the  optical  parts  cannot  be  obtained  in  the  beginning,  it 
is  wise  to  secure  a  stand  upon  which  all  may  be  used  when  they  are  finally 
secured. 

As  to  the  objectives.  The  best  that  can  be  afforded  should  be  obtained. 
Certainly  at  the  present,  the  apochromatics  stand  at  the  head,  although  the  best 
achromatic  objectives  approach  them  very  closely. 

2  133.  Mechanical  Parts  or  Stand. — The  stand  should  be  low  enough  so 
that  it  can  be  used  in  a  vertical  position  on  an  ordinary  table  without  inconven- 
ience; it  should  have  a  jointed  (flexible)  pillar  for  inclination  at  any  angle  to 
the  horizontal.  The  adjustments  for  focusing  should  be  two, — a  coarse  ad- 
justment or  rapid  movement  with  rack  and  pinion,  and  a  fine  adjustment  by 
means  of  a  micrometer  screw.  Both  adjustments  should  move  the  entire  tube 
of  the  microscope.  The  body  or  tube  should  be  short  enough  for  objectives 
corrected  for  the  short  or  160  millimeter  tube-length.  It  is  an  advantage  to 
have  the  draw-tube  graduated  in  centimeters  and  millimeters.  The  lower  end 
of  the  draw  tube  and  of  the  tube  should  each  possess  a  standard  screw  for 
objectives  (frontispiece).  The  stage  should  be  quite  large  for  the  examina- 
tion of  slides  with  serial  sections  and  other  large  objects.  The  substage  fittings 
should  be  so  arranged  as  to  enable  one  to  use  the  condenser  or  to  dispense  en- 
tirely with  diaphragms.  The  condenser  mounting  should  allow  up  and  down 
motion. 

\  134.  Quality  and  Cost.— In  order  that  teachers  and  students  may  get 
a  good  general  idea  of  the  appearance  of  microscopes  of  various  makers  for 
high  school  and  advanced  laboratory  work  a  few  pictures  are  appended  of  the 
microscopes  most  used  in  the  United  States.  This  has  been  rendered  possible 
by  the  courtesy  of  the  manufacturers  or  importers.  The  micrcscopes  are  ar- 
ranged in  alphabetical  order  of  the  makers. 

Laboratory  microscopes  which  will  answer  nearly  all  the  requiremenls  for 
work  in  Biology,  including  Histology,  Embryology,  Pathology  and  Bacteri- 
ology, are  listed  in  the  makers  catalogs  at  about  $75.00.  The  less  expensive  mi- 
croscopes shown  are  listed  at  $25  to  $45.  Fortunately  in  the  State  of  New  York 
the  State  pays  half  for  high  school  apparatus,  so  that  there  is  no  reason  why 
every  high  school  should  not  be  properly  equipped  with  microscopes  of  a  good 
grade.  To  avoid  misunderstanding  it  should  be  added  that  the  quality  of  the 
oculars  and  objectives  on  the  high  school  microscopes  figured  is  the  same  as 


76  LIGHTING  AND  FOCUSIXG  [  CH.  II 

for  the  best  laboratory  microscopes.     The  mechanical  work  also  is  of  excel- 
lent quality. 

During  the  last  few  years  great  vigor  has  been  shown  in  the  microscopical 
world.  This  has  been  stimulated  largely  by  the  activity  in  biological  science 
and  the  widespread  appreciation  of  the  microscope,  not  only  as  a  desirable, 
but  as  a  necessary  instrument  for  study  and  research.  The  production  of  the 
new  kinds  of  glass,  (Jena  glass),  and  the  apochromatic  objectives  has  been  a 
no  less  potent  factor  in  promoting  progress.  The  student  is  advised  to  write 
to  one  or  more  of  the  opticians  for  complete  catalogs.  (See  list,  p.  2  of  cover) . 


STANDARD  SIZES  RECOMMENED  BY  THE  ROYAL 
MICROSCOPICAL  SOCIETY 


$  135.  Society  Screw. — Owing  to  the  lack  of  uniformity  in  screws  for 
microscope  objectives,  the  Royal  Microscopical  Society  of  London,  in  1857, 
made  an  earnest  effort  to  introduce  a  standard  size. 

In  order  to  facilitate  the  introduction  of  this  universal  screw,  or  as  it  soon 
came  to  be  called  "  The  Society  Screw,'"  the  Royal  Microscopical  Society  under- 
took to  supply  standard  taps.  From  the  mechanical  difficulty  in  making  these 
taps  perfect  there  soon  came  to  be  considerable  difference  in  the  "Society 
Screws,"  and  the  object  of  the  society  in  providing  a  universal  screw  was 
partly  defeated.  (See  Edward  Bausch,  Trans.  Amer.  Micr.  Soc.,  1884,  p.  153.) 

In  1884  the  American  Microscopical  Society  appointed  Mr.  Edward  Bausch 
and  Prof.  William  A.  Rogers  upon  a  committee  to  correspond  with  the  Roya] 
Microscopical  Society,  with  a  view  to  perfecting  the  standard  "  Society  Screw," 
or  of  adopting  another  standard  and  of  perfecting  methods  by  which  the  screws 
of  all  makers  might  be  truly  uniform.  Although  this  matter  was  earnestly 
considered  at  the  time  by  the  Royal  Microscopical  Society,  the  mechanical 
difficulties  were  so  great  that  the  improvements  were  abandoned. 

Fortunately,  however,  during  the  year  1896  that  society  again  took  hold 
of  the  matter  in  earnest,  and  the  "  Society  Screw"  is  now  accurate,  and  facili- 
ties for  obtaining  the  standard  are  so  good  that  there  is  a  reasonable  certainty 
that  the  universal  screw  for  microscopic  objectives  may  be  realized.  It  is 
astonishing  to  see  how  widely  the  "  Society  Screw  has  been  adopted.  Indeed 
there  is  not  a  maker  of  first-class  microscopes  in  the  world  who  does  not  supply 
the  objectives  and  stands  with  the  "Society  Screw,"  and  an  objective  in 
England  or  America  which  does  not  have  this  screw  should  be  looked  upon 
with  suspicion.  That  is,  it  is  either  old,  cheap,  or  not  the  product  of  one  of 
the  great  opticians.  For  the  Standard,  or  "  Society  Screw,"  see:  Trans.  Roy. 
Micr.  Soc.,  1857,  pp.  39-41;  1859,  pp.  92-97;  1860,  pp.  103-104.  (All  to  be 
foundin  Quar.  Jour.  Micr.  Sci.,  o.  s.,  vols.VI,  VII,  VIII).  Proc.  Amer.  Micr. 
Soc.  1884,  p.  274;  1886,  p.  199;  1893,  p.  38.  Journal  of  the  Royal  Microscopical 
Society,  August,  1896. 


CH.  //]  LIGHTING  AND  FOCUSING  77 

In  this  last  paper  of  four  pages  the  matter  is  very  carefully  gone  over  and 
full  specifications  of  the  new  screw  given.  It  conforms  almost  exactly  with 
the  original  standard  adopted  by  the  society,  but  means  have  been  devised  by 
which  it  may  be  kept  standard. 

This  paper  is  of  so  much  importance  historically  and  practically  that  it  de- 
serves to  appear  in  every  work  on  the  modern  microscope.  It  is  therefore 
here  repeated  entire  : 

FROM    THE   JOURNAL    OF   THE    ROYAL    MICROSCOPICAL   SCCIETY 

AUGUST,     1896 

"The  Royal  Microscopical  Society's  Standard  Screw-Thread  for  Nose- 
piece  and  Object-Glasses  of  Microscopes." 

"  Being  the  report  of  a  sub-committee  of  the  Council,  drawn  up  by  Conrad 
Beck,  F.R.M.S.,  Secretary  to  the  Sub-Committee.  Read  June  I7th,  1896." 

"The  so-called  Standard  Screw-Thread  of  the  Royal  Microscopical  Society 
has  been  but  an  imperfect  standard,  and  has  not  ensured  that  interchangea- 
bility  which  it  originally  promised.  It  has  been  our  duty  to  investigate  the 
causes  of  this  state  of  affairs,  and  to  formulate  a  plan  by  which  such  an  incon- 
venience should  be  remedied  in  the  futnre. " 

"  Without  going  too  closely  into  the  entire  history  of  the  subject,  we  pro- 
pose to  briefly  explain  the  reasons  why  the  original  standard  was  not  efficient 
for  practical  purposes,  and  then  to  state  the  plan  which  the  Council  of  the 
Royal  Microscopical  Society  has  now  adopted  for  the  future." 

The  specification  of  the  original  standard  screw  was  as  follows  : 

\  136.  Form  of  Thread. — "  Whitworth  thread,  i.  e.,  a  V-shaped  thread, 
sides  of  thread  inclined  at  an  angle  of  55°  to  each  other,  one-sixth  of  the  V 
depth  of  the  thread  being  rounded  off  at  the  top  of  the  thread,  and  one-sixth 
of  the  thread  being  rounded  off  at  the  bottom  of. the  thread." 

"  Pitch  of  Screw,  36  to  the  inch. 

Length  of  Thread  on  Object-Glass,  0.125  in. 

Plain  Fitting  above  Thread  of  Object-Glass  0.15  in.  long,  to  be  about  the 
size  of  the  bottom  of  male  thread. 

Length  of  Thread  of  Nose-Piece  not  less  than  0.125  in. 

Diameter  of  the  Object-Glass  Screw  at  the  bottom  of  the  screw,  0.7626  in. 

Diameter  of  the  Nose-Piece  Screw  at  the  bottom  of  the  thread,  0.8  in." 

"When  the  exact  form  of  the  Whitworth  screw-thread  is  calculated  it  will 
be  found  that  this  allows  a  difference  between  the  male  and  female  screw  of 
0.0018  in.,  which  is  in  itself  quite  sufficient  margin  of  looseness  to  make  an 
easy  fit." 

"  The  society  had  two  plug  and  ring  gauges,  one  0.8  in.,  and  the  other 
o  7626  in.,  made  by  Whitworth  as  standards  for  the  use  of  the  Society,  and  it 
has  been  shown  that  if  an  adjustable  tap  and  die  (as  recommended  by  the  late 
Mr.  Richard  Beck  in  a  paper  printed  in  the  "Transactions  of  the  Microscopi- 
cal Society,"  1859,  P-  92)  be  made  which  could  be  accurately  adjusted  to  these 
standard  sizes  so  that  the  tap  exactly  fitted  the  0.8  in.  ring  size,  and  the  die 


78  LIGHTING  AND  FOCUSING  \CH.  II 

exactly  fitted  the  0.7626  in.  plug,  the  exact  standard  screw  as  originally  sug- 
gested could  be  adhered  to.  These  adjustable  taps  and  dies  were  not  used  for 
cutting  the  thread,  but  for  passing  over  each  thread  after  it  had  been  cut  to 
approximately  the  right  size.  That  this  method  will  work  satisfactorily,  is 
evidenced  by  the  fact  that  in  the  late  Mr.  Richard  Beck's  firm  the  method  has 
been  in  successful  operation  ever  since." 

"The  use,  however,  of  such  a  system  involved  the  necessity  of  every  maker 
being  provided  with  adjustable  tap  and  die,  and  also  the  two  pairs  of  plug  and 
ring  Whitworth  sizes,  together  with  a  means  of  accurately  sharpening  the 
adjustable  tap  and  die.  And  it  was  found  in  practice  that  microscope  makers 
were  not  universally  prepared  to  go  to  such  an  outlay  for  a  matter  which  at 
that  time  did  not  appear  to  be  of  such  importance  as  has  since  proved  to  be 
the  case." 

"Therefore  the  Society  issued  solid  taps,  and  finding  that,  as  is  well  known 
to  be  the  case,  a  solid  tap  could  not  be  made  to  an  exactly  accurate  size  owing 
to  the  alteration  of  the  steel  during  the  process  of  hardening  and  tempering, 
they  had  them  made  somewhat  larger  than  the  standard  0.8  in.  gauge.  An 
additional  reason  for  their  being  larger  was  to  allow  for  the  slight  wearing  of 
the  tap  after  prolonged  use." 

"  Here,  however,  there  was  no  record  of  the  amount  larger  which  the  taps 
were  made,  and  although  the  first  set  appear  to  have  been  carefully  manufac- 
tured, those  which  were  from  time  to  time  obtained  were  less  and  less  like  the 
original,  and  in  this  manner  a  discrepancy  arose  which  the  arrangements  now 
adopted  by  the  Council  are  intended  to  correct  for  the  future." 

"  Beyond  the  fact  that  the  Council  specify  that  the  diameter  of  the  plain 
fitting  of  the  object-glass  should  be  as  near  as  possible  to,  but  not  exceeding 
0.759  in->  and  that  the  length  of  this  fitting  has  been  reduced  to  o. i  in.,  the 
original  specification  of  the  standard  screw  is  only  altered  as  to  the  exact 
diameters  of  the  screw  itself." 

"  The  original  specification  of  these  diameters  allowed  only  0.0018  for 
clearance  between  the  male  and  female  screw." 

"  If  absolutely  exact  sizing  taps  and  dies  could  be  made  which  should  not 
wear,  the  original  diameters  might  have  been  adhered  to,  but  as  has  been  pre- 
viously pointed  out,  adjustable  dies  in  connection  with  gauges,  etc.,  are 
requisite  for  this." 

"  The  Council  has  been  able  to  obtain  taps  and  dies  which  are  guaranteed 
not  to  vary  more  than  i/iooo  of  an  inch  larger  or  smaller  than  the  nominal 
size.  And  they  are  therefore  having  manufactured  a  series  of  taps  of  the 
nominal  diameter  on  the  top  of  the  screw-thread  of  0.8015  in.  which  will  not 
vary  more  than  from  0.8005  in.  to  0.8025  in.  To  insure  this  the  Council  has 
ordered  a  Whitworth  plug  and  ring,  size  0.803  ^n-  ^n  diameter,  and  no  tap  will 
be  allowed  to  be  stamped  with  the  Society's  stamp  unless  it  will  pass  easily 
through  this  0.803  in.  ring,  and  unless  it  is  of  such  a  size  that  it  will  not  enter 
the  0.8  in.  standard  gauge  already  in  the  Society's  possession." 

"They  are  also  having  made  a  series  of  dies  of  the  nominal  inside  diameter 
on  the  top  of  the  thread  of  0.7611  in.,  which  will  not  vary  more  than  from 
0.7601  to  0.7621.  To  test  this  the  Council  has  ordered  a  Whitworth  plug  and 


('//.  77] 


LICHTINC  AND  FOCUSINC 


79 


ring,  size  0.7596  iu.  diameter,  and  no  die  will  be  allowed  to  be  stamped  with 
the  Society's  stamp  unless  it  will  pass  easily  over  the  0.7596  in.  plug  and  will 
not  pass  over  the  0.7626  in.  plug." 

"  These  taps  and  dies  will  be  for  sale  almost  immediately,  at  cost  price, 
2/.  155.  for  each  pair  of  tap  and  dies,  and  it  is  earnestly  requested  that  every 
maker  of  Microscopes  will  possess  himself  of  a  pair  of  these  sizing  gauges." 

"  The  Council  believe  that  at  such  time  as  these  sizing  taps  and  dies  have 
come  into  universal  use  the  standard  screw-thread  will  have  been  put  upon  a 
permanent  basis,  and  complete  interchangeability  of  all  object-glasses  will 
have  been  established." 

3  137.  New  Specification  of  the  Royal  Microscopical  Society  Standard 
Screw. 


FIG.  69 

"Thread. — Whitworth  screw,  i.  e.,a  V-shaped  thread,  sides  of  thread 
inclined  at  an  angle  of  55°  to  each  other,  one-sixth  of  the  V  depth  being 
rounded  off  at  the  top  and  the  bottom  of  the  thread. 

Pitch. — 36  to  the  inch. 

Length  of  Thread  on  Object-Glass  0.125  in- 


So  LIGHTING  AND  FOCUSING  [  CH.  II 

Plain  Fitting  above  Thread  of  Object-Glass  o.  i  in.  long,  not  to  exceed 
0.759  in.  in  diameter. 

Diameter  (C)  of  Thread  on  Object-Glass  at  top  of  thread  not  to  exceed 
o  7982  in.,  or  to  be  less  than  0.7952  in. 

Diameter  (D)  of  Thread  on  Object-Glass  at  bottom  of  thread  not  to 
exceed  0.7626  in.,  or  to  be  less  than  0.7596  in. 

Length  of  Screw  of  Nose-Piece  to  be  not  less  than  0.125  *n- 

Diameter  of  Screw  of  Nose-Piece  (A)  at  top  of  thread  not  to  exceed  0.7674 
in.,  or  be  less  than  0.7644  in. 

Diameter  of  Screw  of  Nose-Piece  (B)  at  bottom  of  thread  not  to  exceed 
0.803  in->  or  De  less  than  o.S  in." 

\  138.  Standard  Size  Oculars  and  Substage  Condensers. — For  a  considera- 
tion of  these,  with  measurements,  see  .$  53,  98. 


MARKERS  AND  MECHANICAL  STAGES 


Markers  are  devices  to  facilitate  the  finding  of  some  object  or  part  which 
it  is  especially  desired  to  refer  to  again  or  to  demonstrate  to  a  class.  The 
mechanical  stage  makes  it  much  easier  to  follow  out  a  series  of  objects,  to 
move  the  slide  when  using  high  powers,  and  for  complete  exploration  of  a 
preparation.  Most  of  the  mechanical  stages  have  scales  or  scales  and  verniers, 
by  which  an  object  once  recorded  may  be  readily  found  again. 

§  139.  Marker  for  Preparations.  (Figs.  70-72). — This  instrument  con. 
sists  of  an  objective-like  attachment  which  may  be  screwed  into  the  nose-piece 
of  the  microscope.  It  bears  on  its  lower  end  a  small  brush  and  the  brush  can 
be  made  more  or  less  eccentric  and  can  be  rotated,  thus  making  a  larger  or 
smaller  circle.  In  using  the  marker  the  brush  is  dipped  in  colored  shellac  or 
other  cement  and  when  the  part  of  the  preparation  to  be  marked  is  found  and 
put  exactly  in  the  middle  of  the  field  the  objective  is  turned  aside  and  the 
marker  turned  into  position.  The  brush  is  brought  carefully  in  contact  with 
the  cover-glass  and  rotated.  This  will  make  a  delicate  ring  of  the  colored 
cement  around  the  object.  Within  this  very  small  area  the  desired  object  can 
be  easily  found  on  any  microscope.  The  brush  of  the  marker  should  be 
cleaned  with  95%  alcohol  after  it  is  used.  (Proc.  Amer.  Micr.  Soc.,  1894,  pp. 
112-118.) 

\  140.  Pointer  in  the  Ocular. — The  Germans  have  a  pointer  ocular  (Spitzen. 
Okular),  an  ocular  with  one  or  two  delicate  rods  or  pointers  at  the  level  of  the 
real  image,  that  is,  at  the  level  of  the  diaphragm  (Figs.  26,  36,  D).  For  the 
purposes  of  demonstrating  any  particular  structure  or  object  in  the  field,  a 
temporary  pointer  may  be  easily  inserted  in  any  ocular  as  follows:  Remove 
the  eye-lens  and  with  a  little  mucilage  or  Canada  Balsam  fasten  a  hair 
from  a  camel's  hair  or  other  fine  brush  to  the  upper  surface  of  the 


CII.  //] 


UCIITIM; 


FOCUSING 


81 


diaphragm  (Fig.  3&D)  so  that  it  will  project  about  half  way  across  the 
opening.  If  one  uses  this  ocular,  the  pointer  will  appear  in  the  field 
and  one  can  place  the  specimen  so  that  the  pointer  indicates  it  exactly, 
as  in  using  a  pointer  on  a  diagram  or  on  the  black-board.  It  is  not  known  to 
the  author  who  devised  this  method.  It  is  certainly  of  the  greatest  advantage 
in  demonstrating  objects  like  amoebas  or  white  blood  corpuscles  to  persons 
not  familiar  with  them,  as  the  field  is  liable  to  have  in  it  many  other  objects 
which  are  more  easily-  seen. 


SS 


70  71 

FIGS.  70-72.     Sectional  Views  of  the  two  Forms  of  the  Marker. 


72 


FIG.  70.  The  simplest  form  of  marker.  It  consists  of  the  part  SS  with 
the  milled  edge  (M) .  This  part  bears  the  society  or  objective  screw  for  attach, 
ing  the  marker  to  the  microscope.  R.  Rotating  part  of  the  marker.  This 
bears  the  eccentric  brush  (B)  at  its  lower  end.  The  brush  is  on  the  wire  (  W], 
This  wire  is  eccentric,  and  may  be  made  more  or  less~5O  by  bending  the  wire. 
The  central  dotted  line  coincides  with  the  axis  of  the  microscope.  The  revolv- 
ing part  is  connected  with  the  "  Society  Screw  "  by  the  small  scrczv  (S). 

FIG.  7r.  SS,  R,  and  B.  All  parts  same  as  with  Fig.  70,  except  that  the 
brush  is  carried  by  a  sliding  cylinder  the  end  view  being  indicated  in  Fig.  72. 

I    141.     Mechanical   Stage. — For  High   School  and   ordinary   laboratory 
work  a  mechanical  stage  is  not  needed;  but  for  much  work,   especially   where 
high  objectives  are  used  a  mechanical  stage  is  of  great  advantage.     It   is  also 
advantageous  if  the  mechanical  stage  can  be  easily  removed. 
The  one  found  on  the  most  expensive  American  and  F,nglish    microscopes  for 
the  last  twenty  years  and  the  one  now  present  on  the  larger  continental  micro- 
scopes, is  excellent  for  high  powers  and  preparations  of  moderate  dimensions, 
but  for  the  study  of  serial  sectons  and  large  sections  or  preparations  in  general 
mechanical  stages  like  those  shown  in  Figs.  79-89  are  more  useful.     This  form 
of  mechanical  stage  has  the  advantage  of  giving  great  lateral  and  forward  and 


82 


LIGHTING  AND  I'OCl  'SING 


[_CH. 


backward  motion.  It  is  a  modification  of  the  mechanical  stage  of  Tolles. 
The  modification  consists  in  doing  away  with  the  thin  plate  and  having  a 
clamp  to  catch  the  ends  of  the  glass  slide.  The  slide  is  then  moved  on  the 
face  of  the  stage  proper.  This  modification  was  first  made  by  Mayall.  It  has 
since  been  modified  by  Reichert,  Zeiss,  L/eitz,  and  others  in  Europe  and  by 
the  Bausch  &  L,omb  Optical  Co.,  Qeeen  &  Co.,  and  the  Spencer  Lens  Co.,  in 
America. — Jour.  Roy.  Micr.  Soc.,  1885,  p.  122.  See  also  Zeit.  Wiss.  Mikro- 
skopie  (II)  1885,  pp.  289-295;  1887  (IV,  pp.  25-30). 

Those  figured  below  have  the  great  advantage  of  ready  removal  from  the 
stage  of  the  microscope,  thus  leaving  it  free.  They  have  also  the  very  excel- 
lent feature  that  with  them  one  can  explore  an  entire  slide  full  of  serial  sec- 
tions, as  the  sections  are  ordinarily  mounted,  i.e. ,  under  a  cover-glass  24X50 
mm. 


73 


74 


FIGS.  73-75.     Specimens  Showing  the  Use  of  the  Marker. 

In  Fig:  73  a  section  of  a  series  is  marked  to  indicate  that  this  section  shozvs 
something  especially  well.  In  Fig.  74  some  blood  corpuscles  showing  ingested 
carbon  very  satisfactorily  are  surrounded  by  a  minute  ring,  and  in  Fig.  75  the 
lines  of  a  micrometer  are  ringed  to  facilitate  finding  the  lines. 


CH.  //] 


LAHORA  TORY  MICROSCOPES 


FIG.  76.     The  Bausch  &  Loinb  Optical  Co's 
Detachable  Mechanical  Stage. 


FIG.  77.     The  Detachable  Mechani- 
cal Stage  of  Lcitz. 


FIG.  78.     The  Spencer  Lens  Co's  Detachable  Mechanical 
Stage  of  Great  Range. 


LA nOKA  TOR } '  MICROSCOPES  [  CH.\II 


FIG.  79.     The  Bausch  &  Lomb  Optical  Go's  AVer  Model  DDH  Microscope^ 


Cll.  II  ]  LA  'iOKA  '/'( >A' } '  Ml(  'R(  >.SO  1PES  85 


:FIG.  80.     The  Bausch  &  Lomb  Optical  Co's  Microscope  BH.     Handle  Type. 


86  LABORATORY  .MICROSCOPES  \_CH.I1 


FIG.  81.     The  Bausch  &  Lomb  Optical  Co's  Microscope  B  without  Handle^ 


r//.  //] 


L.l/H  IRA  TORY  MICROSCi  1PES 


FIG.  82.     The  Bausch  &  Lomb  Optical  Co's  Model  A  //  Microscope,  Handle 

Type.     The  coarse  adjustment  is  by  a  sliding  tube,  and  the 

pillar  is  not  jointed.     See  also  Fig.  140  on  p.  178. 


88 


LABORATORY  MICROSCOPES 


[  CH.  II 


FIG.  83.     Beck's  London  Microscope,  Regents  Model,  icith  Handle 
and  New  Fine  Adjustment.     See  also  Fig.  755. 


Cf/.  //] 


I.  A  HORA  T(  ^R  } '   MICR(  >St  'OPES 


FIG.  Leitz  Universal  Microscope,  Stand  A  with  Large  Tube  and 

Special  Fine  Adjustment.     See  also  Figs.  142,  150. 


LABORATORY  MICROSCOPES 


[  CH.  II 


FIGS.  85,  86.  Queen  &  Co's  Continental  Microscope,  No.  II.  Dust- 
proof,  triple  nose-piece.  The  difference  between  this  and  the  ordinary  form 
can  be  seen  by  comparing  with  Fig.  87.  This  form  of  revolving  nose-piece  has 
been  made  for  many  years  by  Winkel  of  Goettingen.  See  legend  of  Fig.  40. 


CH.  //] 


LA  BORA  TOR ) '  MICROSCOPES 


FIG.  88.     Reicherfs  Laboratory  Microscope  with  Handle.     This  handle 
is  so  attached  that  it  does  not  preclude  the  ordinary  means  for  fine  adjustment. 


92 


LA  BORA  TOR  } '  MICR  OSCOPES 


[  CH.  II 


FIG.  89.     The  Spencer  Lens  Co's  New  Model  No.  10  Microscope 
(Specially  for  Photo-Micrography, 


CH.  //] 


LABOKA  TOR ) '  MICROSa  >/'/-:S 


93 


FIG.  90.     The  Spencer  Lens  Co's  ^ficroscope  No.  40  with  curved  Spring  inside 
the  Arm  and  Pillar  so  that  they  may  be  safely  used  as  a  Handle. 


94 


LABORATORY  MICROSCOPES 


L  CH.  II 


FIG.  91.     7~^  Spencer  Lens  Co's  Microscope  No.  36  with  an  extra  large  Stage. 


CH.  II] 


LABOR  A  TOR  Y  MICROSCOPES 


95 


FIG.  92.     The  Spencer  Lens   Co's  Microscope  No.   70  with 
double  Nose-Piece  and  no    Condenser. 


LABORATORY  MICROSCOPES 


[  CH.  II 


FIG.  93.      Voigtlander  &  Sohn's  Laboratory  Microscope  No.  IV. 
For  their  "large  Stand,  see  Fig.  //<?. 


CH.  //] 


L  A  HORA  TOR  Y  MICROSCOPES 


97 


FIG  94.  Zeiss  Microscope  /"  with  Mechanical  Stage.  This  figure  from 
Zeiss'  Catalog  No.  30,  represents  the  Continental  Model  of  Microscope  in  its 
most  perfect  form. 

K.     Milled  head  of  the  screw  for  the  lateral  movements  of  the  stage. 

L.  Screw  for  fiLving  the  laterally  moving  mechanism  of  the  stage.  By 
unscrewing  this  the  laterally  moving  part  may  be  removed,  leaving  the  plain 
stage. 

W.     Screw  for  moving  the  stage  forward  and  backward. 


CH.  U] 


LA  BORA  TOR  Y  MICROSCOPES 


98 


FIG.  95.     Zeiss  Stand  /°  for  Photo-Micrography 


CHAPTER  III 


INTERPRETATION  OF  APPEARANCES 


APPARATUS    AND    MATERIAL    FOR    CHAPTER    III 

A  laboratory,  compound  microscope  (I  132);  Preparation  of  fly's  wing,  50 
per  cent  glycerin;  Slides  and  covers;  Preparation  of  letters  in  stairs  (Fig.  96). 
Mucilage  for  air-bubbles  and  olive  or  clove  oil  for  oil-globules  (§  149-152). 
Solid  glass  rod,  and  glass  tube  (?  157-159);  Collodion  ($  159);  Carmine,  India 
ink,  or  lamp  black  (?  161-163);  Frog,  castor  oil  and  micro-polariscope  ($  164). 

INTERPRETATION   OF   APPEARANCES   UNDER   THE   MICROSCOPE 

>5  142.  General  Remarks. — The  experiments  in  this  chapter 
are  given  secondarily  for  drill  in  manipulation,  but  primarily  so  that 
the  student  may  not  be  led  into  error  or  be  puzzled  by  appearances 
which  are  constantly  met  with  in  microscopical  investigation.  Any- 
one can  look  into  a  microscope,  but  it  is  quite  another  matter  to  in- 
terpret correctly  the  meaning  of  the  appearances  seen. 

It  is  especially  important  to  remember  that  the  more  of  the 
relations  of  any  object  are  known,  the  truer  is  the  comprehension  of 
the  object.  In  microscopical  investigation  every  object  should  be 
scrutinized  from  all  sides  and  under  all  conditions  in  which  it  is 
likely  to  occur  in  nature  and  in  microscopical  investigation.  It  is 
best  also  to  begin  with  objects  of  considerable  size  whose  character 
is  well  known,  to  look  at  them  carefully  with  the  unaided  eye  so  as 
to  see  them  as  wholes  and  in  their  natural  setting;  then  a  low  power 
is  used,  and  so  on,  step  by  step  until  the  highest  power  available 
has  been  employed.  One  will  in  this  way  see  less  and  less  of  the 
object  as  a  whole,  but  every  increase  in  magnification  will  give  in- 
creased prominence  to  detail,  detail  which  might  be  meaningless 
when  taken  alone  and  independent  of  the  object  as  a  whole.  The 
pertinence -of  this  advice  will  be  appreciated  when  the  student  under- 
takes to  solve  the  problems  of  histology;  for  even  after  all  the  years 
of  incessant  labor  spent  in  trying  to  make  out  the  structure  of  man 


ioo  INTERPRETATION  OF  APPEARANCES        \_CH.II1 

and  the  lower  animals,  many  details  are  still  in  doubt,  the  same 
visual  appearances  being  quite  differently  interpreted  by  eminent 
observers. 

Appearances  which  seem  perfectly  unmistakable  with  a  low 
power  may  be  found  erroneous  or  very  inadequate,  for  details  of  struc- 
ture that  were  undistinguishable  with  the  low  power  may  become 
perfectly  evident  with  a  higher  power  or  a  more  perfect  objective. 
Indeed  the  problems  of  microscopic  structure  appear  to  become  ever 
more  complex,  for  difficulties  overcome  by  improvements  in  the 
microscope  simply  give  place  to  new  difficulties,  which  in  some  cases 
render  the  subject  more  obscure  than  it  appeared  to  be  with  the  less 
perfect  appliances. 

The  need  of  the  most  careful  observation  and  constant  watchful- 
ness lest  the  appearances  may  be  deceptive  are  thus  admirably  stated 
by  Dallinger  (see  Carpenter-Dallinger,  p.  427):  ;'The  correctness 
of  the  conclusions  which  the  microscopist  will  draw  regarding  the 
nature  of  any  object  from  the  visual  appearances  which  it  presents 
to  him  when  examined  in  the  various  modes  now  specified  will 
necessarily  depend  in  a  great  degree  upon  his  previous  experience 
in  microscopic  observation  and  upon  his  knowledge  of  the  class  of 
bodies  to  which  the  particular  specimen  may  belong.  Not  only  are 
observations  of  any  kind  liable  to  certain  fallacies  arising  out  of  the 
previous  notions  which  the  observer  may  entertain  in  regard  to  the- 
constitution  of  the  objects  or  the  nature  of  the  actions  to  which  his 
attention  is  directed,  but  even  the  most  practiced  observer  is  apt  to 
take  no  note  of  such  phenomena  as  his  mind  is  not  prepared  to  ap- 
preciate. Errors  and  imperfections  of  this  kind  can  only  be  cor- 
rected, it  is  obvious,  by  general  advance  in  scientific  knowledge; 
but  the  history  of  them" affords  a  useful  warning  against  hasty  con- 
clusions drawn  from  a  too  cursory  examination.  If  the  history  of 
almost  any  scientific  investigation  were  fully  made  known  it  would 
generally  appear  that  the  stability  and  completeness  of  the  conclu- 
sions finally  arrived  at  had  been  only  attained  after  many  modifica- 
tions, or  even  entire  alterations,  of  doctrine.  And  it  is  therefore  of 
such  great  importance  as  to  be  almost  essential  to  the  correctness  of 
our  conclusions  that  they  should  not  be  finally  formed  and  announced 
until  they  have  been  tested  in  every  conceivable  mode..  It  is  due 
to  science  that  it  should  be  burdened  with  as  few  false  facts  [artifacts] 
and  false  doctrines  as  possible.  It  is  due  to  other  truth- seekers 


<•//.///]        INTERPRETATION  OF  APPEARANCES  101 

that  they  should  not  be  misled,  to  the  great  waste  of  their  time  and 
pains,  by  our  errors.  And  it  is  due  to  ourselves  that  we  should  not 
commit  our  reputation  to  the  chance  of  impairment  by  the  premature 
formation  and  publication  of  conclusions  which  may  be  at  once  re- 
versed by  other  observers  better  informed  than  ourselves,  or  may  be 
proved  fallacious  at  some  future  time,  perhaps  even  by  our  own 
more  extended  and  careful  researches.  The  suspension  of  the  judg- 
ment whenever  there  seems  room  for  doubt  is  a  lesson  inculcated  by  all 
those  philosophers  who  have  gained  the  highest  repute  for  practical 
wisdom;  and  it  is  one  which  the  microscopist  cannot  too  soon  learn 
or  too  constantly  practice." 

For  these  experiments  no  condenser  is  to  be  used  except  where 
specifically  indicated. 

§  143.  Dust  or  Cloudiness  on  the  Ocular. — Employ  the  16 
mm.  (-.;  in.)  objective,  low  ocular,  and  fly's  wing  as  object. 

Unscrew  the  field-lens  and  put  some  particles  of  lint  from  dark 
cloth  on  its  upper  surface.  Replace  the  field-lens  and  put  the  ocu- 
lar in  position  (§  55).  Light  the  field  well  and  focus  sharply.  The 
image  will  be  clear,  but  part  of  the  field  will  be  obscured  by  the  ir- 
regular outline  of  the  particles  of  lint.  Move  the  object  to  make 
sure  this  appearance  is  not  due  to  it. 

Grasp  the  ocular  by  the  milled  ring,  just  above  the  tube  of  the 
microscope,  and  rotate  it.  The  irregular  objects  will  rotate  with  the 
ocular.  Cloudiness  or  particles  of  dust  on  any  part  of  the  ocular 
may  be  detected  in  this  way. 

§  144.  Dust  or  Cloudiness  on  the  Objective. — Employ  the 
same  ocular  and  objective  as  before  and  the  fly's  wing  as  object. 
Focus  and  light  well,  and  observe  carefully  the  appearance.  Rub 
glycerin  on  one  side  of  a  slide  near  the  end.  Hold  the  clean  side  of 
this  end  close  against  the  objective.  The  image  will  be  obscured* 
and  cannot  be  made  clear  by  focusing.  Then  use  a  clean  slide  and 
the  image  may  be  made  clear  by  elevating  the  tube  slightly.  The 
obscurity  produced  in  this  way  is  like  that  caused  by  clouding  the 
front-lens  of  the  objective.  Dust  would  make  a  dark  patch  on  the 
image  that  would  remain  stationary  while  the  object  or  ocular  is 
moved. 

If  a  small  diaphragm  is  employed  and  it  is  close  to  the  object, 
only  the  central  part  of  the  field  will  be  illuminated,  and  around  the 


102  INTERPRETATION  OF  APPEARANCES        [CII.1I1 

small  light  circle  will  be  seen  a  dark  ring  (Fig.  49).  If  the  dia- 
phragm is  lowered  or  a  sufficiently  large  one  employed  the  entire 
field  will  be  lighted. 

§145.  Relative  Position  of  Objects  or  parts  of  the  same 
object.  The  general  rule  is  that  objects  highest  up  come  into  focus 
last  in  focusing  up,  first  in  focusing  down. 

§  146.  Objects  having  Plane  or  Irregular  Outlines. — As 
object  use  three  printed  letters  in  stairs  mounted  in  Canada  balsam 
(Fig.  96).  The  first  letter  is  placed  directly  upon  the  slide,  and 
covered  with  a  small  piece  of  glass  about  as  thick  as  a  slide.  The 
second  letter  is  placed  upon  this  and  covered  in  like  manner.  The 
third  letter  is  placed  upon  the  second  thick  cover  and  covered  with 
an  ordinary  cover-glass.  The  letters  should  be  as  near  together  as 
possible,  but  not  over-lapping.  Employ  the  same  ocular  and  objec- 
tive as  above  (§143). 

a  FIG.    96.       Letters     mounted     in 

b  stairs  to  show  the  order  of  coming 

into  focus. 

a,  b,  c,  d.       The    various    letters 
indicated  by  the  oblique  roiv  of  black 

marks  in  sectional  view.  Slide.  The  glass  slide  on  which  the  letters  are 
mounted. 

Lower  the  tube  till  the  objective  almost  touches  the  top  letter, 
then  look  into  the  microscope,  and  slowly  focus  up.  The  lowest 
letter  will  first  appear  and  then,  as  it  disappears,  the  middle  one  will 
appear,  and  so  on.  Focus  down,  and  the  top  letter  will  first  appear, 
then  the  middle  one,  etc.  The  relative  position  of  objects  is  deter- 
mined exactly  in  this  way  in  practical  work. 

For  example,  if  one  has  a  micrometer  ruled  on  a  cover-glass  15- 
25  hundredths  mm.  thick,  it  is  not  easy  to  determine  with  the  naked 
eye  which  is  the  ruled  surface.  But  if  one  puts  the  micrometer 
under  a  microscope  and  uses  a  3  mm.  (^  in.)  objective,  it  is  easily 
determined.  The  cover  should  be  laid  on  a  slide  and  focused  till 
the  lines  are  sharp.  Now,  without  changing  the  focus  in  the  least 
turn  the  cover  over.  If  it  is  necessary  to  focus  up  to  get  the  lines 
of  the  micrometer  sharp,  the  lines  are  on  the  upper  side.  If  one 
must  focus  down,  the  lines  are  on  the  under  surface.  With  a  thin 
cover  and  delicate  lines  this  method  of  determining  the  position  of 
the  rulings  is  of  considerable  practical  importance. 


(//.///]         INTERPRETATION  OF  APPEARANCES  103 

«?  147.  Determination  of  the  Form  of  Objects. — The  pro- 
cedure is  exactly  as  for  the  determination  of  the  form  of  large  ob- 
jects. That  is,  one  must  examine  the  various  aspects.  For  ex- 
ample, if  one  were  placed  in  front  of  a  wall  of  some  kind  he  could 
not  tell  whether  it  was  a  simple  wall  or  whether  it  was  one  side  of  a 
building  unless  in  some  way  he  could  see  more  than  the  face  of  the 
wall.  In  other  words,  in  order  to  get  a  correct  notion  of  any  body, 
one  must  examine  more  than  one  dimension, — two  for  plane  sur- 
faces, three  for  solids.  So  for  microscopic  objects,  one  must  in  some 
way  examine  more  than  one  face.  To  do  this  with  small  bodies  in 
a  liquid  the  bodies  may  be  made  to  roll  over  by  pressing  on  one 
edge  of  the  cover-glass.  And  in  rolling  over  the  various  aspects  are 
presented  to  the  observer.  With  solid  bodies,  like  the  various 
organs,  correct  notions  of  the  form  of  the  elements  can  be  deter- 
mined by  studying  sections  cut  at  right  angles  to  each  other.  The 
methods  of  getting  the  elements  to  roll  over,  and  of  sectioning  in 
different  planes  are  inconstant  use  in  Histology,  and  the  microscopist 
who  neglects  to  see  all  sides  of  the  tissue  elements  has  a  very  inade- 
quate and  often  a  very  erroneous  conception  of  their  true  form. 

§  148.  Transparent  Objects  having  Curved  Outlines.— 
The  success  of  these  experiments  will  depend  entirely  upon  the  care 
and  skill  used  in  preparing  the  objects,  in  lighting,  and  in  focusing. 

Employ  a  3  mm.  (>6  in.)  or  higher  objective  and  a  high  ocular 
for  all  the  experiments.  It  may  be  necessary  to  shade  the  object 
(§  120)  to  get  satisfactory  results.  When  a  diaphragm  is  used  the 
opening  should  be  small  and  it  should  be  close  to  the  object. 

§  149.  Air  Bubbles. — Prepare  these  by  placing  a  drop  ot  thin 
mucilage  on  the  center  of  a  slide  and  beating  it  with  a  scalpel  blade 
until  the  mucilage  looks  milky  from  the  inclusion  of  air  bubbles. 
Put  on  a  cover-glass  but  do  not  press  it  down. 

FIG.  97.  Diagram 
showing  how  to  place  a 
cover-glass  upon  an  ob- 
ject  with  the  forceps. 

§  150.  Air  Bubbles  with  Central  Illumination. — Shade  the 
object;  and  with  the  plane  mirror,  light  the  field  with  central  light 
(Fig.  28). 


104 


INTERPRETATION  OF  APPEARANCES        [  CH.  Ill 


Search  the  preparation  until  an  air  bubble  is  found  appearing 
about  i  mm.  in  diameter,  get  it  into  the  center  of  the  field,  and  if 
the  light  is  central  the  air  bubble  will  appear  with  a  wide,  dark,  cir- 
cular margin  and  a  small  bright  center.  If  the  bright  spot  is  not  in 
the  center,  adjust  the  mirror  until  it  is. 

This  is  one  of  the  simplest  and  surest  methods  of  telling  when 
the  light  is  central  or  axial  when  no  condenser  is  used  (§  74). 

Focus  both  up  and  down,  noting  that,  in  focusing  up,  the  cen- 
tral spot  becomes  very  clear  and  the  black  ring  very  sharp.  On 
elevating  the  tube  of  the  microscope  still  more  the  center  becomes 
dim,  and  the  whole  bubble  loses  its  sharpness  of  outline. 

§  151.  Air  Bubbles  with  Oblique  Illumination. — Remove 
the  sub-stage  of  the  microscope  and  all  the  diaphragms.  Swing  the 
mirror  so  that  the  rays  may  be  sent  very  obliquely  upon  the  object 
(Fig.  28,  C).  The  bright  spot  will  appear  no  longer  in  the  center 
but  on  the  side  away  from  the  mirror  (Fig.  98,  A). 

$  152.  Oil  Globules. — Prepare  these  by  beating  a  small  drop 
of  clove  oil  with  mucilage  on  a  slide  and  covering  as  directed  for 
air  bubbles  (§  150),  or  use  a  drop  of  milk. 

§  153.  Oil  Globules  with  Central  Illumination. — Use  the 
same  diaphragm  and  light  as  above  (§  150).  Find  an  oil  globule 
appearing  about  i  mm.  in  diameter.  If  the  light  is  central  a  bright 
spot  will  appear  in  the  center  as  with  air.  Focus  up  and  down  as 
with  air,  and  note  that  the  bright  center  of  the  oil  globules  is  clear- 
est last  in  focusing  up. 


FIG.  98.  Very  small  Globules  of  Oil  (O)  and  an  Air  Bub- 
bles (A)  seen  by  Oblique  Light.  Surface  vietc.  The  arrow 
indicates  the  direction  of  the  light  rays. 


154.     Oil  Globules  with  Oblique  Illumination.— Remove 
the  sub-stage,  etc.,  as  above,  and  swing  the  mirror  to  one  side  and 


CH.  ///]        INTERPRETATION  OF  APPEARANCES  105 

light    with   oblique    light.     The  bright  spot  will  be  eccentric,  and 
will  appear  to  be  on  the  same  side  as  the  mirror  (Fig.  98,0). 

S  155-  Oil  and  Air  Together. — Make  a  preparation  exactly 
as  described  for  air  bubbles  (§  149),  and  add  at  one  edge  a  little  of 
the  mixture  of  oil  and  mucilage  (§  152);  cover  and  examine. 

The  sub-stage  need  not  be  used  in  this  experiment.  Search 
the  preparation  until  an  air  bubble  and  an  oil  globule,  each  ap- 
pearing about  i  mm.  in  diameter,  are  found  in  the  same  field  of 
view.  Light  first  with  central  light,  and  note  that,  in  focusing  up, 
the  air  bubble  comes  into  focus  first  and  that  the  central  spot  is 
smaller  than  that  of  the  oil  globule.  Then,  of  course,  the  black 
ring  will  be  wider  in  the  air  bubble  than  in  the  oil  globule.  Make 
the  light  oblique.  The  bright  spot  in  the  air  bubble  will  move 
i*  «'<*}' from  the  mirror  while  that  in  the  oil  globule  will  move  toward 
it.  See  Fig.  91.* 

£  156.  Air  and  Oil  by  Reflected  Light. — Cover  the  dia- 
phragm or  mirror  so  that  no  transmitted  light  (§  73)  can  reach  the 
preparation,  using  the  same  preparation  as  in  §  155.  The  oil  and 
air  will  appear  like  globules  of  silver  on  a  dark  ground.  The  part 
that  was  darkest  in  each  with  transmitted  light  will  be  lighted,  and 
the  bright  central  spot  will  be  somewhat  dark.t 

§  157.  Distinctness  of  Outline. — In  refraction  images  this 
depends  on  the  difference  between  the  refractive  power  of  a  body 
and  that  of  the  medium  which  surrounds  it.  The  oil  and  air  were 
very  distinct  in  outline  as  both  differ  greatly  in  refractive  power 
from  the  medium  which  surrounds  them,  the  oil  being  more  refrac- 
tive than  the  mucilage  and  the  air  less.  (Figs.  61-63.) 

Place  a  fragment  of  a  cover- glass  on  a  clean  slide,  and  cover  it 

*  It  should  be  remembered  that  the  image  in  the  compound  microscope  is 
inverted  (Fig.  26),  hence  the  bright  spot  really  moves  toward  the  mirror  for 
air,  and  away  from  it  for  oil. 

f  It  is  possible  to  distinguish  oil  and  air  optically,  as  described  above,  only 
when  quite  high  powers  are  used  and  very  small  bubbles  are  selected  for  ob- 
servation. If  a  16  mm.  (  23  in. )  is  used  instead  of  a  3  mm.  (%  in. )  objective, 
the  appearances  will  vary  considerably  from  that  given  above  for  the  higher 
power.  It  is  well  to  use  a  low  as  well  as  a  high  power.  Marked  differences 
will  also  be  seen  in  the  appearances  with  objectives  of  small  and  of  large 
aperture. 


io6 


INTERPRE  TA  TION  OF  APPEARANCES        [  CH.  Ill 


(see  under  mounting).  The  outline  will  be  distinct  with  the  un- 
aided eye.  Use  it  as  object  and  employ  the  16  mm.  (23  in.)  objec- 
tive and  high  ocular.  Light  with  central  light.  The  fragment 
will  be  outlined  by  a  dark  band.  Put  a  drop  of  water  at  the  edge 
of  the  cover-glass.  It  will  run  in  and  immerse  the  fragment.  The 
outline  will  remain  distinct,  but  the  dark  band  will  be  somewhat 
narrower.  Remove  the  cover-glass,  wipe  it  dry,  and  wipe  the  frag- 
ment and  slide  dry  also.  Put  a  drop  of  50%  glycerin  on  the  middle 
of  the  slide  and  mount  the  fragment  of  cover-  glass  in  that.  The 
dark  contour  will  be  much  narrower  than  before. 

FIG.  99.  Section  of  an  air  bub- 
ble and  an  oil  globule  in  water  (H2O). 
The  air  bubble  although  spherical  in 
form  gives  only  a  virtual  focus,  indi- 
cated by  the  dotted  lines  below  the  bub- 
ble. As  it  is  surrounded  by  a  denser 
medium  it  acts  like  a  concave  lens  in 
air  (Fig.  10}  .  The  focus  of  the  oil 
globule  is  real  as  it  is  denser  than  the 
surrounding  medium.  Axis,  —  the 
principal  axis.  F,  principal  focus.  It 

is  virtual  and  below  for  the  air  bubble  ;  real  and  above  for  the  oil  globule. 

H^O.     Water  or  a  mixture  of  water  and  gum  arabic  serving  as  a  mounting 

medium  (\ 


Draw  a  solid  glass  rod  out  to  a  fine  thread.  Mount  one  piece 
in  air,  and  the  other  in  50%"  glycerin.  Put  a  cover-glass  on  each. 
Employ  the  same  optical  arrangement  as  before.  Examine  the  one 
in  air  first.  There  will  be  seen  a  narrow,  bright  band,  with  a  wide, 
dark  band  on  each  side  (Fig.  100,  a;. 

FIG.  100.  Solid  glass  rod  showing 
the    appearance    when    viewed    with 
transmitted,  central  light,  and  with  an 
objective  of  medium  aperture. 
a.     Mounted  in  air.     b.     Mounted  in  50  per  cent  glycerin. 

The  one  in  glycerin  will  show  a  much  wider  bright  central 
band,  with  the  dark  borders  correspondingly  narrow  (Fig.  100,  b). 
The  dark  contour  depends  also  on  the  numerical  aperture  of  the 
objective  —  being  wider  with  low  apertures.  This  can  be  readily 
understood  when  it  is  remembered  that  the  greater  the  aperture  the 
more  oblique  the  rays  of  light  that  can  be  received,  and  the  dark 


CH.  Ill}        INTERPRETATION  ()/•'  Al'PEARANCES  107 

band  simply  represents  an  area  in  which  the  rays  are  so  greatly  bent 
or  refracted  (Figs.  61-63)  tna*  they  cannot  enter  the  objective  and 
contribute  to  the  formation  of  the  image  ;  the  edges  are  dark  sim- 
ply because  no  light  from  them  reaches  the  observer. 

If  the  glass  rod  or  any  other  object  were  mounted  in  a  medium 
of  the  same  color  and  refractive  power,  it  could  not  be  distinguished 
from  the  medium.  * 

A  very  striking  and  satisfactory  demonstration  may  be  made 
by  painting  a  zone  or  band  of  eosin  or  other  transparent  color  on  a 
solid  glass  rod,  and  immersing  the  rod  in  a  test  tube  or  vial  of  cedar 
oil,  clove  oil  or  turpentine.  Above  the  liquid  the  glass  rod  is  very 
evident,  as  it  is  also  at  the  colored  zone,  but  at  other  levels  it  can 
hardly  be  seen  in  the  liquid. 

£  158.  Highly  Refractive. — This  expression  is  often  used  in 
describing  microscopic  objects,  (medullated  nerve  fibers,  for  ex- 
ample), and  means  that  object  will  appear  to  be  bordered  by  a  wide, 
dark  margin  when  it  is  viewed  by  transmitted  .light.  And  from  the 
above  (§  157),  it  would  be  known  that  the  refractive  power  of  the 
object,  and  the  medium  in  which  it  was  mounted  must  differ  con- 
siderably. 

FIG.  10 1.  Solid  glass  rod  coated 
with  collodion  to  show  a  double  con- 
tour. Toward  one  end  the  collodion 
had  gathered  in  a  fusiform  drop. 


§  159.  Doubly  Contoured. — This  means  that  the  object  is 
bounded  by  two,  usually  parallel  dark  lines  with  a  lighter  band  be- 
tween them.  In  other  words,  the  object  is  bordered  by  (i)  a  dark 
line,  (2)  a  light  band,  and  (3)  a  second  dark  line  (Fig.  101). 

This  may  be  demonstrated  by  coating  a  fine  glass  rod  (§  157) 
with  one  or  more  coats  of  collodion  or  celloidin  and  allowing  it  to 
dry,  and  then  mounting  in  50%  glycerin  as  above.  Employ  a  3 
mm. (y&  in.)  or  higher  objective,  light  with  transmitted  light,  and 
it  will  be  seen  that  where  the  glycerin  touches  the  collodion  coating 

*  Some  of  the  rods  have  air  bubbles  in  them,  and  then  there  results  a 
capillary  tube  when  they  are  drawn  out.  It  is  well  to  draw  out  a  glass  tube 
into  a  fine  thread  and  examine  it  as  described.  The  central  cavity  makes  the 
experiment  much  more  complex. 


io8  INTERPRETATION  OF  APPEARANCES        [  CM.  Ill 

there  is  a  dark  line — next  this  is  a  light  band,  and  finally  there  is  a 

second  dark  line  where  the  collodion  is  in  contact   with   the  glass 
rod.*     (Fig.    101). 

$  160.  Optional  Section. — This  is  the  appearance  obtained 
in  examining  transparent  or  nearly  transparent  objects  with  a 
microscope  when  some  plane  below  the  upper  surface  of  the  object 
is  in  focus.  The  upper  part  of  the  object  which  is  out  of  focus 
obscures  the  image  but  slightly.  By  changing  the  position  of  the 
objective  or  object,  a  different  plane  will  be  in  focus  and  a  different 
optical  section  obtained.  The  most  satisfactory  optical  sections  are 
obtained  with  high  objectives  having  large  aperture. 

Nearly  all  the  transparent  objects  studied  may  be  viewed  in 
optical  section.  A  striking  example  will  be  found  in  studying 
mammalian  red  blood-corpuscles  on  edge.  The  experiments  with 
the  solid  glass  rods  (Fig.  ioo)furnish  excellent  and  striking  examples 
of  optical  sections. 

§  161.  Currents  in  Liquids. — Employ  the  16  mm.  (7310.) 
objective,  and  as  object  put  a  few  particles  of  carmine  on  the  middle 
of  a  slide,  and  add  a  drop  of  water.  Grind  the  carmine  well  with  a 
scalpel  blade,  and  then  cover  it.  If  the  microscope  is  inclined,  a 
current  will  be  produced  in  the  water,  and  the  particles  of  carmine 
will  be  carried  along  by  it.  Note  that  the  particles  seem  to  flow  up 
instead  of  down — why  is  this  ? 

Lamp-black  rubbed  in  water  containing  a  little  mucilage  answers 
well  for  this  experiment. 

§  162.  Velocity  Under  the  Microscope. — In  studying  cur- 
rents or  the  movement  of  living  things  under  the  microscope,  one 
should  not  forget  that  the  apparent  velocity  is  as  unlike  the  real 
velocity  as  the  apparent  size  is  unlike  the  real  size.  If  one  consults 
Fig.  42  it  will  be  seen  that  the  actual  size  of  the  field  of  the  micro- 
sc9pe  with  the  different  objectives  and  oculars  is  inversely  as  the 
magnification.  That  is,  with  great  magnification  only  a  small  area 
can  be  seen.  The  field  appears  to  be  large,  however,  and  if  any 


*  The  collodion  used  is  a  6%  solution  of  gun  cotton  in  equal  parts  of  sul- 
phuric ether  and  95%  alcohol.  It  is  well  to  dip  the  rod  two  or  three  times  in 
the  collodion  and  to  hold  it  vertically  while  drying.  The  collodion  will  gather 
in  drops,  and  one  will  see  the  difference  between  a  thick  and  a  thin. membran- 
ous covering  (Fig.  101). 


CH.  ///]         INTERPRETATION  OF  APPEARANCES  109 

object  moves  across  the  field  it  may  appear  to  move  with  great 
rapidity,  whereas  if  one  measures  the  actual  distance  passed  and 
notes  the  time,  it  will  be  seen  that  the  actual  motion  is  quite  slow. 
One  should  keep  this  in  mind  in  studying  the  circulation  of  the 
blood.  The  truth  of  what  has  just  been  said  can  be  easily  demon- 
strated in  studying  the  circulation  in  the  gills  of  Necturus,  or  in  the 
frog's  foot,  by  using  first  a  low  power  in  which  the  field  is  actually 
of  considerable  diameter  (Fig.  42,  Table,  §  58)  and  then  using  a  high 
power.  With  the  high  power  the  apparent  motion  will  appear  much 
more  rapid.  For  spiral,  serpentine  and  other  forms  of  motion,  see 
Carpenter-Dallinger,  p.  433. 

£  163.  Pedesis  or  Brownian  Movement. — Employ  the 
same  object  as  above,  but  a  3  mm.  (l/3  in.)  or  higher  objective  in 
place  of  the  16  mm.  Make  the  body  of  the  microscope  vertical,  so 
that  there  may  be  no  currents  produced.  Use  a  small  diaphragm 
and  light  the  field  well.  Focus  and  there  will  be  seen  in  the  field 
large  motionless  masses,  and  between  them  small  masses  in  constant 
motion.  This  is  an  indfinite,  dancing  or  oscillating  motion. 

This  indefinite  but  continuous  motion  of  small  particles  in  a 
liquid  is  called  Pe-d'e'  sis  or  Brownian  movement.  Also,  but  im- 
properly, molecular  movement,  from  the  smallness  of  the  particles. 

The  motion  is  increased  by  adding  a  little  gum  arabic  solution 
or  a  slight  amount  of  silicate  of  soda  or  soap;  sulphuric  acid  and 
various  saline  compounds  retard  or  check  the  motion.  One  of  the 
best  objects  is  lamp-black  ground  up  with  a  little  gum  arabic.  Car- 
mine prepared  in  the  same  way,  or  simply  in  water,  is  excellent; 
and  very  finely  powdered  pumice-stone  in  water  has  for  many  years 
been  a  favorite  object. 

Pedesis  is  exhibited  by  all  solid  matter  if  it  is  finely  enough 
divided  and  in  a  suitable  liquid.  In  the  minds  of  most,  no  adequate 
explanation  has  yet  been  offered. 

Compare  the  pedetic  motion  with  that  of  a  current  by  slightly 
inclining  the  tube  of  the  microscope.  The  small  particles  will  con- 
tinue their  independent  leaping  movements  while  they  are  carried 
along  by  the  current.  The  pedetic  motion  makes  it  difficult  to  ob- 
tain good  photographs  of  milk  gobules  and  other  small  particles. 
The  difficulty  may  be  overcome  by  mixing  the  milk  with  a  very 
weak  solution  of  gelatin  and  allowing  it  to  cool  (see  Ch.IX). 


no  INTERPRETATION  OF  APPEARANCES        [C//.  Ill 

§  164.  Demonstration  of  Pedesis  with  the  Polarizing 
Microscope. — (Ch.  VI.)  The  following  demonstration  shows  con- 
clusively that  the  pedetic  motion  is  real  and  not  illusive.  (Ranvier, 

P-  I73-) 

Open  the  abdomen  of  a  dead  frog  (an  alcoholic  or  formalin 
specimen  is  satisfactory).  Turn  the  viscera  to  one  side  and  observe 
the  small,  whitish  masses  at  the  emergence  of  the  spinal  nerves. 
With  fine  forceps  remove  one  of  these  and  place  it  on  the  middle  of 
a  clean  slide.  Add  a  drop  of  water,  or  of  water  containing  a  little 
gum  arabic.  Rub  the  white  mass  around  in  the  drop  of  liquid  and 
soon  the  liquid  will  have  a  milky  appearance.  Remove  the  white 
mass,  place  a  cover-glass  on  the  milky  liquid  and  seal  the  cover  by 
painting  a  ring  of  castor  oil  all  around  it,  half  the  ring  being  on  the 
slide  and  half  on  the  cover-glass.  This  is  to  avoid  the  production 
of  currents  by  evaporation. 

Put  the  preparation  under  the  microscope  and  examine  with,  first 
a  low  power  then  a  high  power  (3  mm.  or  y%  in.).  In  the  field  will 
be  seen  multitudes  of  crystals  of  carbonate  of  lime;  the  larger  crys- 
tals are  motionless  but  the  smallest  ones  exhibit  marked  pedetic 
movement. 

Use  the  micro-polariscope,  light  with  great  care  and  exclude  all 
adventitious  light  from  the  microscope  b}-  shading  the  object  (§  120) 
and  also  by  shading  the  eye.  Focus  sharply  and  observe  the  pedetic 
motion  of  the  small  particles,  then  cross  the  polarizer  and  analyzer, 
that  is,  turn  one  or  the  other  until  the  field  is  dark.  Part  of  the 
large  motionless  crystals  will  shine  continuously  and  a  part  will  re- 
main dark,  but  small  crystals  between  the  large  ones  will  shine  for 
an  instant,  then  disappear,  only  to  appear  again  the  next  instant. 
This  demonstration  is  believed  to  furnish  absolute  proof  that  the 
pedetic  movement  is  real  and  not  illusory. 

§  165.  Muscae  Volitantes. — These  specks  or  filaments  in  the 
eyes  due  to  minute  shreds  or  opacities  of  the  vitreous  sometimes  ap- 
pear as  part  of  the  object  as  they  are  projected  into  the  field  of 
vision.  They  may  be  seen  by  looking  into  the  well  lighted  micro- 
scope when  there  is  no  object  under  the  microscope.  They  may  also 
be  seen  by  looking  at  brightly  illuminated  snow  or  other  white  sur- 
face. By  studying  them  carefully  it  will  be  seen  that  they  are  some- 
what movable  and  float  across  the  field  of  vision,  and  thus  do  not 
remain  in  one  position  as  do  the  objects  under  observation.  Further- 


CH.  II I~\        INTERPRETATION  OF  APPEARANCES  in 

more,  one  may,  by  taking  a  little  pains,  familiarize  himself  with  the 
special  forms  in  his  own  eyes  so  that  the  more  conspicuous  at  least 
may  be  instantly  recognized. 

£  1 66.  Miscellaneous  Observations. — In  addition  to  the 
above  experiments  it  is  very  strongly  recommended  that  the  student 
follow  the  advice  of  Beale,  p.  248,  and  examine  first  with  a  low 
then  with  a  higher  power,  mounted  dry,  then  in  water,  lighted  with 
reflected  light,  then  with  transmitted  light,  the  following:  Potato, 
wheat,  rice  and  corn  starch,  easily  obtained  by  scraping  the  potato 
and  the  grains  mentioned;  bread  crumbs;  portions  of  feather.  Por- 
tions of  feather  accidentally  present  in  histological  preparations 
have  been  mistaken  for  lymphatic  vessels  (Beale,  288).  Fibers  of 
cotton,  linen  and  silk.  Textile  fibers  accidentally  present  have  been 
considered  nerve  fibers,  etc.  Human  and  animal  hairs.  Study 
with  especial  care  hairs  from  various  parts  of  the  body  of  the  animals 
used  for  dissection  in  the  laboratory  where  you  work.  These  are 
liable  to  be  present  in  histological  preparations,  and  unless  their 
character  is  understood  there  is  chance  for  much  confusion  and 
erroneous  interpretation.  The  scales  of  butterflies  and  moths,  es- 
pecially the  common  clothes  moths.  The  dust  swept  from  carpeted 
and  wood  floors.  Tea  leaves  and  coffee  grounds.  Dust  found  in 
living  rooms  and  places  not  frequently  dusted.  In  the  last  will  be 
found  a  regular  museum  of  objects. 

If  it  is  necessary  to  see  all  sides  of  an  ordinary  gross  object, 
and  to  observe  it  with  varying  illumination  and  under  various  con- 
ditions of  temperature,  moisture,  etc.,  in  order  to  obtain  a  fairly  ac- 
curate and  satisfactor)7  knowledge  of  it,  so  much  the  more  is  it 
necessary  not  to  be  satisfied  it  microscopical  observation  until  every 
means  of  investigation  and  verification  has  been  called  into  service, 
and  then  of  the  image  that  falls  upon  the  retina,  only  such  details 
will  be  noted  as  the  brain  behind  the  eye  is  ready  to  appreciate. 

§  167.  Summary  for  Proper  Interpretation. — To  summar- 
ize this  chapter  and  leave  with  the  beginning  student  the  result  of 
the  experience  of  many  eminent  workers: 

1.  Get    all    the    information   possible  with    the  unaided  eye. 
See  the  whole  object  and  all  sides  of  it,  so  far  as  possible. 

2.  Examine   the    preparation  with  a  simple  microscope  in  the 
same  thorough  way  for  additional  detail. 


H2  INTERPRETATION  OF  APPEARANCES        [  CH.  Ill 

3.  Use  alow  power  of  the  compound  microscope. 

4.  Use  a  higher  power. 

5.  Use  the  highest  power  available  and  applicable.     In    this 
way  one  sees  the  object  as  a  whole  and  progressively  more  and  more 
details.     Then  as  the  object  is  viewed  from   two  or  more  aspects, 
something  like  a  correct   notion    may   be  gained    of   its   form    and 
structure. 

§  1 68.  Zeiss-Greenough  Binocular,  Erecting  Micro- 
scope.— As  shown  in  figure  102  this  consists  of  a  microscope  stage 
with  two  tubes  mounted  side  by  side  and  moving  on  the  same  rack 
and  pinion.  Either  tube  can  be  used  without  the  other.  The  ocu- 
lars are  capable  of  greater  or  less  separation  to  suit  the  eyes  of 
different  observers.  In  the  large  cylinder  near  the  top  is  placed  a 
Porro  prism  which  erects  the  image.  This  microscope  gives  most 
perfect  stereoscopic  images  and  also  erect  ones,  and  therefore  is  es- 
pecially adapted  for  dissection  and  for  studying  objects  of  consider- 
able thickness,  like  injected  preparations  etc.  It  is  interesting  to 
note  that  the  binocular  microscope  constructed  by  Cherubin 
D'Orleans,  1677,  was  composed  in  like  manner  of  two  microscopes 
side  by  side.  It  of  course  had  no  erecting  prisms  (For  statement 
and  figure  of  this  early  binocular,  see  Mayall,  p.  17,  18). 

§  169.  Wenham's  Binocular  Microscope. — This  is  illus- 
trated in  Figs.  103-104.  There  is  but  a  single  objective.  The  light 
from  this  is  divided  by  a  prism,  a  part  of  it  passing  to  the  right  and 
a  part  to  the  left  eye.  That  to  the  right  eye  passes  directly,  that  to 
the  left  is  twice  internally  reflected  by  the  prism  to  give  it  the  right 
inclination. 

In  order  to  get  the  stereoscopic  effect  with  the  binocular  there 
must  be  an  image  in  both  eyes,  and  to  ensure  this  the  oculars  must 
be  separable  so  that  the  eye- points  are  the  same  width  as  the  pupils 
of  the  eyes  of  the  observer. 

One  can  tell  whether  there  is  binocular  vision  in  his  first  trials 
by  closing  first  one  eye  and  then  the  other.  If  an  image  is  seen 
without  moving  the  head  whichever  eye  is  closed  then  of  course 
both  eyes  are  seeing  an  image  and  one  should  get  the  appearance  of 
relief  characteristic  of  stereoscopic  images.  If  one  does  not  see  with 
both  eyes  the  eye- points  are  too  close  or  too  far  seperated  for  his 
pupils.  The  tubes  should  be  seperated  or  approximated  until  each 


I'll.   Ill]         INTERPRETATION  OF  APPEARANCES  113 

eye  sees  the  image.     After  one  is  nsed  to  the  stereoscopic  appear- 
ance when  seeing  with  both  eyes  he  can  tell  instantly  whether  the 


FIG.  102.  Greenough's  Erecting  Binocular  Microscope.  This  consists  of 
tti'o  microscope  tubes  mounted  side  by  side.  The  oculars  may  be  approximated 
or  separated  for  the  eyes  of  different  observers.  The  images  are  erected  by  the 
Porro  prisms  in  the  large  rounded  part  of  the  tube.  (Zeiss'  Catalog.) 


114 


INTERPRETATION  OF  APPEARANCES        [  Cff.  Ill 


binocular    is    properly    adjusted    for    his   eyes.       (See  Carpeuter- 
Dallinger  for  fuller  discussion  of  Binoculars.) 


103  104 

FIG.  103.  Sectional  View  of  Wenham's  Binocular  Microscope,  a.  The 
prism  which  extends  partly  across  the  field  and  directs  about  half  of  the  light 
to  the  left  eye  (L).  A  part  of  the  light  extends  directly  to  the  right  eye  (R). 
c,  b.  Field  lenses  of  the  right  and  the  left  oculars. 

FIG.  104.  An  enlargement  of  the  Prism  used  in  the  Wenham  Binocular 
Microscope,  a,  b,  c,  d  Represent  the  course  of  the  ray  for  the  left  eye.  It  is 
internally  reflected  at  the  points  b,  c,  and  given  the  proper  direction  to  enter 
the  left  eye. 

REFERENCES    FOR    CHAPTER    III. 

For  general  discussions  :  Carpenter-Dallinger,  A.  E.  Wright,  Principles  of 
Microscopy,  Ch.  V.;  Beale  ;  Spitta,  Microscopy,  Ch.  xviii.;  Beck's  Cantor 
Lectures,  lect.  IV. 

For  pedesis  see  Carpenter-Dallinger,  p.  431  ;  Beale,  p.  195  ;  Jevons  in 
Quart.  Jour.  Science,  n.  s.,  Vol.  VIII  (1878),  p.  167. 

For  the  original  account  of  this  see  Robert  Brown,  "  Botanical  appendix 
to  Captain  King's  voyage  to  Australia,"  Vol.  II,  p.  534  (1826). 

See  also  Dr.  C.  Aug.  Sigm.  Schultze,  "  Mikroskopische  Untersuchungen 


O/.  ///]        INTERPRETATION  OF  APPEARANCES  115 

iiber  des  Herren  Robert  Brown  Entdeckung  lebender,  selbst  im  Feuer  unzer- 
storbarer  Theilchen  in  alien  Korpern."  From  "Die  Gesellschaft  fiir  Befor- 
derung  der  Xaturwissenchaften  zu  Frieburg."  (1828.) 

For  overcoming  pedesis  for  photography  see  Gage,  The  use  of  a  solution 
of  gelatin  to  obviate  pedesis  in  photographing  milk  globules  and  other  minute 
objects  in  water.  Transactions  Amer.  Micr.  Soc.,  Vol.  XXIV.,  1902,  p.  21. 

For  figures  (photo-micrographs,  etc.  )  of  the  various  forms  of  starch,  see 
Bulletin  No.  13  of  the  Chemical  Division  of  the  U.  S.  Department  of  Agri- 
culture. For  Hair  and  Wool,  see  Bulletin  of  the  National  Association  of  Wool 
Growers,  1875,  p.  470,  Proc.  Amer.  Micr.  Soc.,  1884,  pp.  65-68.  Herzfeld, 
translated  by  Salter. — The  technical  testing  of  yarns  and  textile  fabrics,  Lon- 
don, 1898.  See  also  the  Bibliography  at  the  end  for  works  relating  to  adulter- 
ation of  foods,  etc.,  for  further  discussions  of  the  elements  used  in  foods  and 
drugs. 

For  different  appearances  due  to  the  illuminator,  see  Nelson,  in  Jour.  Roy. 
Micr.  Soc.,  1891,  pp.  90-105  ;  and  for  the  illusory  appearances  due  to  diffrac- 
tion phenomena,  see  Carpenter-Dallinger,  p.  434.  Mercer.  Trans.  Amer.  Micr. 
Soc.,  pp.  321-396.  Also,  A.  E.  Wright's  Principles  of  Microscopy;  Conrad" 
Beck. 

For  the  Binocular  see  Carpenter-Dallinger  ;  Mayall  ;  Spitta. 


1.  Positive  ocular. 

2.  Draw-tube. 

3.  Main  tube  or  body. 

4-5.  Society  screws  in  the  draw-tube  and  body. 

6.  Objective  in  position. 

7.  Stage. 

8.  Spring  for  holding  slides. 

9.  Sub-stage  condenser. 
10.  Iris  diaphragm. 

n.  Plane  and  concave  mirror.  * 

12.  Horse-shoe  base. 

13.  Rack  and  pinion  for  condenser. 

14.  Flexible  pilar. 

15.  vSpiral  spring  of  fine  adjustment. 

16.  Fine  adjustment. 

17.  Coarse  adjustment. 


THE  HICROSCOPE  IN  SECTION 


CHAPTER  IV 


MAGNIFICATION  AND  MICROMETRY 


APPARATUS    AND    MATERIAL    FOR    THIS    CHAPTER 

Simple  and  compound  microscope  (f  172,  174);  Steel  scale  or  rule  divided 
to  millimeters  and  4;  Block  for  magnifier  and  compound  microscope  (\  172, 
176);  Dividers  (\  172,  176);  Stage  micrometer  (|  175);  Wollaston  camera  lucida 
($  176);  Ocular  screw-micrometers  (Figs.  118-120);  Abbe  camera  lucida  (Fig. 
'114).  Necturus  red  blood  corpuscles  (\  184).  Eikonometer  (\  195). 

§  170.  The  Magnification,  Amplification  or  Magnifying 
Power  of  a  simple  or  compound  microscope  is  the  ratio  between 
the  real  and  the  apparent  size  of  the  object  examined.  The  appar- 
ent size  is  obtained  by  measuring  the  virtual  image  (Figs.  26,  43). 
For  determining  magnification  the  object  must  be  of  known  length 
and  is  designated  a  micrometer  (§175).  In  practice  a  virtual  image 
is  measured  by  the  aid  of  some  form  of  camera  lucida  (Figs.  108, 
114),  or  by  double  vision  (§  172).  As  the  length  of  the  object  is 
known,  the  magnification  is  easily  determined  by  dividing  the 
apparent  size  of  the  image  by  the  actual  size  of  the  object.  For 
example,  if  the  virtual  image  measures  40  mm.  and  the  object  mag- 
nified, 2  mm.,  the  amplification  is  40-^-2=20,  that  is,  the  appar- 
ent size  is  20  fold  greater  than  the  real  size. 

Magnification  is  expressed  in  diameters  or  times  linear,  that  is 
but  one  dimension  is  considered.  In  giving  a  scale  at  which  a  micro- 
scopical or  histological  drawing  is  made,  the  word  magnification  is 
frequently  indicated  by  the  sign  of  multiplication  thus  :  X  450,  upon 
a  drawing  means  that  the  figure  or  drawing  is  450  times  as  large  as 
the  object. 

§171.  Magnification  of  Real  Images. — In  this  case  the 
magnification  is  the  ratio  between  the  size  of  the  real  image  and  the 
size  of  the  object,  and  the  size  of  the  real  image  can  be  measured 
directly.  By  recalling  the  work  on  the  function  of  an  objective 


CH.  IV]  MAGNIFICATION  AND  MICROMETRY  117 

(§  60),  it  will  be  remembered  that  it  forms  a  real  image  on  the 
ground  glass  placed  on  the  top  of  the  tube,  and  that  this  real  image 
could  be  looked  at  with  the  eye  or  measurered  as  if  it  were  an  actual 
object.  For  example,  suppose  the  object  were  three  millimeters 
long  and  its  image  on  the  ground  glass  measured  15  mm.,  then  the 
magnification  is  i5-=-3=5,  that  is,  the  real  image  is  5  times  as 
long  as  the  object.  The  real  images  seen  in  photography  are 
mostly  smaller  than  the  objects,  but  the  magnification  is  designated 
in  the  same  way  by  dividing  the  size  of  the  real  image  measured  on 
the  ground  glass  by  the  size  of  the  object.  For  example,  if  the  ob- 
ject is  400  millimeters  long  and  its  image  on  the  ground  glass  is  25 
mm.  long  the  ratio  is  25-7-400— y1^.  That  is,  the  image  is  y1^  as 
long  as  the  object  and  is  not  magnified  but  reduced.  In  marking 
negatives,  as  with  drawings,  the  sign  of  multiplication  is  put  before 
the  ratio,  and  in  the  example  the  designation  is  XT^.  In  photog- 
raphy (Ch.  VIII)  and  when  using  the  magic  lantern  and  the  pro- 
jection microscope  the  images  are  real,  and  may  be  measured  on  the 
screen  as  if  real  pictures. 

MAGNIFICATION    OF    A   SIMPLE    MICROSCOPE 

§  172.     The  Magnification  of  a  Simple  Microscope  is  the 
ratio  between  the  object  magnified  (Fig.  16,  A1!?'),  and  the  virtual 


FIG.  105.     Tripod  Magnifier 

image  (A3B3).  To  obtain  the  size  of  this  virtual  image  place  the 
tripod  magnifier  near  the  edge  of  a  support  of  such  a  height  that 
the  distance  from  the  upper  surface  of  the  magnifier  to  the  table  is 
250  millimeters. 


ii8  MAGNIFICATION  AND  MICROMETRY  \_CH.IV 

As  object,  place  a  scale  of  some  kind  ruled  in  millimeters  on 
the  support  under  the  magnifier.  Put  some  white  paper  on  the 
table  at  the  base  of  the  support  and  on  the  side  facing  the  light. 


FIG.   106.     Ten  Centimeter  Rule.     The  upper  edge  is  divided  into  milli- 
meters, the  lower  into  centimeters  at  the  left  and  half  centimeters  at  the  right. 

Close  one  eye,  and  hold  the  head  so  that  the  other  will  be  near 
the  upper  surface  of  the  lens.  Focus  if  necessary  to  make  the 
image  clear  (§  12).  Open  the  closed  eye  and  the  image  of  the  rule 
will  appear  as  if  on  the  paper  at  the  base  of  the  support.  Hold  the 
head  very  still,  and  with  dividers  get  the  distance  between  any  two 
lines  of  the  image.  This  is  the  so-called  method  of  double  vision 
in  which  the  microscope  image  is  seen  with  one  eye  and  the  dividers 
with  the  others,  the  two  images  appearing  to  be  fused  in  a  single 
visual  field. 

§  173.  Measuring  the  Spread  of  Dividers. — Thisshouldbe 
done  on  a  steel  scale  divided  to  millimeters  and  \. 

As  i  mm.  cannot  be  seen  plainly  by  the  unaided  eye,  place  one 
arm  of  the  dividers  at  a  centimeter  line,  and  with  the  tripod  magni- 
fier count  the  number  of  spaces  on  the  rule  included  between  the 
points  of  the  dividers.  The  magnifier  simply  makes  it  easy  to 
count  the  spaces  on  the  rule  included  between  the  points  of  the 
dividers — it  does  not,  of  course,  increase  the  number  of  spaces  or 
change  their  value. 

As  the  distance  between  any  two  lines  of  the  image  of  the  scale 
gives  the  size  of  the  virtual  image  (Fig.  16,  A3  Bs),  and  as  the  size 
of  the  object  is  known,  the  magnification  is  determined  by  dividing 
the  size  of  the  image  by  the  size  of  the  object.  Thus,  suppose  the 
distance  between  the  two  lines  of  the  image  is  measured  by  the 
dividers  and  found  on  the  steel  scale  to  be  15  millimeters,  and  the 
actual  size  of  the  space  between  the  two  lines  of  the  object  is  2  mil- 
limeters, then  the  magnification  is  15-5-2=7^,  that  is  the  image 
is  7 Y?,  times  as  long  or  wide  as  the  object.  In  this  case  the  image 
is  said  to  be  magnified  7^  diameters,  or  7^  times  linear. 


CH.  II7}  MACXIF1CATION  AND  M1CROMETRY  119 

The  magnification  of  any  simple  magnifier  may  be  determined 
experimentally  in  the  way  described  for  the  tripod  ;  but  this  methcd 
is  of  course  only  possible  when  the  observer  has  two  good  eyes.  If 
he  has  but  one  eye  then  the  magnification  ma}'  be  determined  by 
the  aid  of  a  camera  lucida  (§  176)  or  the  eikouometer  (§  196). 

MAGNIFICATION    OF    A    COMPOUND    MICROSCOPE 

§  174.  The  Magnification  of  a  Compound  Microscope  is 
the  ratio  between  the  final  or  virtual  image  (Fig.  26,  B3A3),  and 
the  object  magnified  (A  B). 

The  determination  of  the  magnification  of  a  compound  micro- 
scope may  be  made  as  with  a  simple  microscope  (§  172),  but  this  is 
fatiguing  and  unsatisfactory. 

§  175.  Stage,  Object  or  Objective  Micrometer. — For  de- 
termining the  magnification  of  a  compound  microscope  and  for  the 
purpose  of  micrometry,  it  is  necessary  to  have  a  finely  divided  scale 
or  rule  on  glass  or  on  metal.  Such  a  finely  divided  scale  is  called  a 
micrometer,  and  for  ordinary  work  one  mounted  on  a  glass  slide 
( i  X  3  in. ,  25  x  76mm. )  is  most  convenient. 

The  spaces  between  the  lines  should  be  y1^  and  y^  mm.  (or  if 
in  inches,  y^  and  TTJVj7  in.).  Micrometers  are  sometimes  ruled  on 
the  slide,  but  more  satisfactorily  on  a  cover-glass  of  known  thick- 
ness, preferably  0.15 — o.  18  mm.  The  covers  should  be  perfectly 
clean  before  the  ruling,  and  afterwards  simply  dusted  off  with  a 
camel's  hair  duster,  and  then  mounted,  lines  downward  over  a  shel- 
lac or  other  good  cell.  (See  Ch.  VII.)  If  one  rubs  the  lines  the 
edges  of  the  furrow  made  by  the  diamond  are  liable  to  be  rounded 
and  the  sharpness  of  the  micrometer  is  lost.  If  the  lines  are  on  the 
slide  and  uncovered  one  cannot  use  the  micrometer  with  an  oil  im- 
mersion, as  the  oil  obliterates  the  lines.  Cleaning  the  slide  makes 
the  lines  less  sharp  as  stated.  If  the  lines  are  coarse,  it  is  an 
advantage  to  fill  them  with  plumbago.  This  may  be  done  with 
some  very  fine  plumbago  on  the  end  of  a  soft  cork,  or  by  using  a 
soft  lead  pencil.  Lines  properly  filled  may  be  covered  with  balsam 
and  a  cover-glass  as  in  ordinary  balsam  mounting  (Ch.  VII). 

£  1-6.  Determination  of  Magnification. — This  is  most 
readily  accomplished  by  the  use  of  some  form  of  camera  lucida 
(Ch.  V),  that  of  Wollaston  being  most  convenient  as  it  may  be 


120  MAGNIFICATION  AND  MICROMETRY  \_CH.Il' 

used  for  all  powers,  and  the  determination  of  the  standard  distance 
of  250  millimeters  at  which  to  measure  the  images  is  readily  deter- 
mined (Fig.  108,  §  178). 

Employ  the  16  mm.  (23  in.)  objective  and  a  37  mm.  (orx8 
ocular  with  a  stage  micrometer  as  object.  For  this  power  the  TV 
mm.  spaces  of  the  micrometer  should  be  used  as  object.  Focus 
sharply. 


FIG.  107.     Abbess  Test  Plate  to  show  the  enclosure  of  the  micrometer  lines 
by  small  rings.     See  also  Fig.  75. 

It  is  somewhat  difficult  to  find  the  micrometer  lines.  To  avoid 
this  it  is  well  to  have  a  small  ring  enclosing  some  of  the  micrometer 
lines  (Fig.  107).  The  light  must  also  be  carefully  regulated.  If 
too  much  light  is  used,  i.  e.,  too  large  an  aperture,  the  lines  will  be 
drowned  in  the  light.  In  focusing  with  the  high  powers  be  very 
careful.  Remember  the  micrometers  are  expensive,  and  one  can- 
not afford  to  break  them.  As  suggested  in  §  83,  focus  on  the  edge 
of  the  cement  ring  enclosing  the  lines,  then  in  focusing  down  to  find 
the  lines,  move  the  preparation  very  slightly,  back  and  forth. 

After  the  lines  are  sharply  focused,  and  the  slide  clamped  in 
position  make  the  tube  of  the  microscope  horizontal,  by  bending  the 
flexible  pillar,  being  careful  not  to  bring  any  strain  upon  the  fine 
adjustment  (frontispiece). 

Put  a  Wollaston  camera  lucida  (Fig.  108  and  Ch.  V)  in  posi- 
tion, and  turn  the  ocular  around  if  necessary  so  that  the  broad  flat 
surface  may  face  directly  upward,  as  shown  in  the  figure.  Elevate 
the  microscope  by  putting  a  block  under  the  base,  so  that  the  per- 
pendicular distance  from  the  upper  surface  of  the  camera  lucida  to 
the  table  is  250  mm.  (§  178).  Place  some  white  paper  on  the 
work-table  beneath  the  camera  lucida. 

Close  one  eye,  and  hold  the  head  so  that  the  other  may  be  very 
close  to  the  camera  lucida.  Look  directly  down.  The  image  will 


C/f.  1 V  ]  MA  GNIFICA  TION  A  ND  MICRO  ME  TR  Y 


121 


appear  to  be  on  the  table.  It  may  be  necessary  to  readjust  the  focus 
after  the  camera  lucida  is  in  position.  If  there  is  difficulty  in  seeing 
dividers  and  image  consult  Ch.  V.  Measure  the  image  with  dividers 
and  obtain  the  power  exactly  as  above  (§  172-173). 

FIG.  108.  Wollaston's  Camera 
Lucida,  showing  the  rays  from 
the  microscope  and  from  the  draw- 
ing surface,  also  the  position  of 
the  pupil  of  the  eye. 

A.vis,  A.vis.  A. vial  rays 
from  the  microscope  and  from  the 
draiving  surface  (  Ch.  V). 

Camera  Lucida.  A  section  of 
the  quadrangular  prism  showing 
the  course  of  the  rays  in  the  prism 
from  the  microscope  to  tlie  eye. 
As  the  rays  are  twice  reflected, 
they  have  the  same  relation  on 
entering  the  eye  that  they  would 
have,  by  looking  directly  into  the 
ocular. 

A.  B.  The  late  ml  rays  from 
the  microscope  and  their  projection 
upon  the  drawing  surface. 


108 


C.  D.     Rays  from  the  drawing  surface  to  the  eye. 

A.  D  A'  D' .  Overlapping  portions  of  the  two  fields,  where  both  the 
microscopic  image  and  the  drawing  surface,  pencil,  etc.,  can  be  seen.  It  is  rep- 
resented by  the  shaded  part  of  the  overlapping  circles  at  the  right. 

Ocular.     The  ocular  of  the  microscope. 

P.     The  drawing  pencil.     Its  point  is  shown  in  the  overlapping  fields. 

Thus:  Suppose  two  of  the  y1^  mm.,  spaces  were  taken  as  object, 
and  the  image  is  measured  by  the  dividers,  and  the  spread  of  the 
dividers  is  found  on  the  steel  rule  to  be  gf  millimeters.  If  the  ob- 
ject is  y'jj  of  a  millimeter  and  the  magnified  image  is  9^  millimeters, 
the  magnification  (which  is  the  ratio  between  size  of  object  and 
image)  is  9 5. -=--^=4.7.  That  is,  the  magnificatfon  is  47  diameters, 
or  47  times  linear.  If  the  fractional  numbers  in  the  above  example 
trouble  the  student,  both  may  be  reduced  to  the  same  denomination, 
thus:  If.  the  size  of  the  image  is  found  to  be  g-f  mm.  this  number 
may  be  reduced  to  tenths  mm.,  so  it  will  be  of  the  same  denomina- 
tion as  the  object.  In  9  mm.  there  are  90  tenths,  and  in  f  there 
are  4  tenths,  then  the  whole  length  of  the  image  is  90+4=94  tenths 


MA GNIFICA TION  AND  MICROME TRY  [ CH. 


of  a  millimeter.  The  object  is  2  tenths  of  a  millimeter,  then  there 
must  have  been  a  magnification  of  94-7-2=47  diameters  in  order  to 
produce  an  image  94  tenths  of  a  millimeter  long. 

Image 


Object- 


FIG.  109  Fig.  no 

Fig.  109-110.  Figures  showing  that  the  size  of  object  and  image  rarv 
directly  as  their  distance  from  the  center  of  the  lens.  In  Fig.  no  one  can  also 
see  why  it  is  necessary  to  focus  down,  i.  e.  bring  the  object  and  objectives  nearer 
together  ivhen  the  tube  is  lengthened.  See  also  Fig.  66. 

Put  the  25  mm.  (i  in.,  C,  or  X  12)  ocular  in  place  of  one  of  37 
mm.  focus,  and  then  put  the  camera  lucida  in  position.  .  Measure 
the  size  of  the  image  with  dividers  and  a  rule  as  before.  The  power 
will  be  considerably  greater  than  when  the  low  ocular  was  used. 
This  is  because  the  virtual  image  (Fig.  26,  B3  A3)  seen  with  the 


CIL  IV}  MAGNIFICATION  AND  MIC&OMETRY  123 

high  ocular  is  larger  than  the  one  seen  with  the  low  one.  The  real 
image  (Fig.  26  A1  B1)  remains  nearly  the  same,  and  would  be  just 
the  same  if  positive,  par-focal  oculars  (§  43,  82,  note)  were  used. 

Lengthen  the  tube  of  the  microscope  50-60  mm.  by  pulling  out 
the  draw-tube.  Remove  the  camera  lucida,  and  focus,  then  replace 
the  camera  and  obtain  the  magnification.  It  is  greater  than  with 
the  shorter  tube.  This  is  because  the  real  image  (Fig.  no)  is 
formed  farther  from  the  objective  when  the  tube  is  lengthened,  and 
the  objective  must  be  brought  nearer  the  object.  The  law  is:  The 
size  of  object  and  image  varies  directly  as  their  distance  from  the  center 
of  the  lens.  The  truth  of  this  statement  is  illustrated  by  Figs.  109 
and  1 10. 

§  177.  Varying  the  Magnification  of  a  Compound  Micro- 
scope.— It  is  seen  from  the  above  experiments  (§176)  that  in- 
.dependently  of  the  distance  at  which  the  microscopic  image  is 
measured  (§  178),  there  are  three  ways  of  varying  the  power  of  a 
compound  microscope.  These  are  named  below  in  the  order  of 
desirability. 

(1)  By  using  a  higher  or  lower  objective. 

(2)  By  using  a  higher  or  lower  ocular. 

(3)  By  lengthening  or  shortening  the  tube  of  the  microscope  (Fig. 
no).* 

§  178.  Standard  Distance  of  250  Millimeters  at  which  the 
Virtual  Image  is  Measured. — For  obtaining  the  magnification  of 
both  the  simple  and  the  compound  microscope  the  directions  were 
to  measure  the  virtual  image  at  a  distance  of  250  millimeters.  This 
is  not  that  the  image  could  not  be  seen  and  measured  at  any  other 
distance,  but  because  some  standard  must  be  selected,  and  this  is 
the  most  common  one.  The  necessity  for  the  adoption  of  some  com- 
mon standard  will  be  seen  at  a  glance  in  Fig.  in,  where  is  repre- 


*Amplifier.  — In  addition  to  the  methods  of  varying  the  magnification 
given  in  \  177,  the  magnification  is  sometimes  increased  by  the  use  of  an 
amplifier,  that  is  a  diverging  lens  or  combination  placed  between  the  objec- 
tive and  ocular  and  serving  to  give  the  image-forming  rays  from  the  objective 
an  increased  divergence.  An  effective  form  of  this  accessory  was  made  by 
Tolles,  who  made  it  as  a  small  achromatic  concavo-convex  lens  to  be  screwed 
into  the  lower  end  of  the  draw-tube  (frontispiece)  and  thus  but  a  short  distance 
above  the  objective.  The  divergence  given  to  the  rays  increases  the  si/.e  of 
the  real  image  about  two-fold. 


124 


MA  GNIFICA  TION  AND  Ml  CROAT E  TR  Y          [  C77.  /  V 


sented  graphically  the  fact  that  the  size  of  the  virtual  image  de- 
pends directly  on  the  distance  at  which  it  is  projected,  and  this  size 
is  directly  proportional  to  the  vertical  distance  from  the  apex  of  the 


FIG.  in.  Figure  show- 
ing the  position  of  the  mi- 
croscope, the  camera  lucida, 
the  eye,  and  the  difference  in 
size  of  the  image  depending 
upon  the  distance  at  which 
it  is  projected  from  from  the 
eye.  (a)  The  size  at  25  on.; 
(b)  at 35  cm.,  (\  i78). 


FIG.  112.  Wollastori 's  camera  lu- 
cida in  position  on  the  upper  end  of  the 
tube  of  the  microscope .  (Cut  loaned  by 
the  Spencer  Lens  Co.) 


FIG.  113.  Simple  microscope  mechanically  supported  by  a  lens  holder. 
One  may  obtain  the  magnifying  power  of  a  simple  microscope  by  the  use  of  a 
camera  lucida  as  with  the  compound  microscope.  (Cut  loaned  by  the  Spen- 
cer Lens  Co. ) 


MAGNIFICATION  AM)  MICROMKTRY 


125 


triangle,  of  which  it  forms  a  base.  The  distance  of  250  millimeters 
has  been  chosen  on  the  supposition  that  it  is  the  distance  of  most 
distinct  vision  for  the  normal  human  eye. 

Demonstrate  the  difference  in  magnification  due  to  the  distance 
at  which  the  image  is  projected,  by  raising  the  microscope  so  that 
the  distance  will  be  350  millimeters,  then  lowering  to  150  milli- 
meters. 


FIG.  114.  Sectional 
view  of  the  Abbe  Cam- 
era Lucida  to  show  that 
in  measuring  the  stand- 
ard distance  of  250  mill- 
imeters, one  must  meas- 
ure along  the  axis  from 
the  point  P,  at  the  left 
of  t 'he prism,  to  the  mir- 
ror, and  from  the  mir- 
ror to  the  drawing  sur- 
face. For  a  full  e.r- 
planation  of  this  camera 
lucida,  see  next  chapter. 


In  preparing  drawings  it  is  often  of  great  convenience  to  make 
them  at  a  distance  somewhat  less  or  somewhat  greater  than  the 
standard.  In  such  a  case  the  magnification  must  be  determined 
for  the  special  distance.  (See  the  next  chapter,  §  207.) 

For  discussion  of  the  magnification  of  the  microscope,  see:  Beale, 
pp.  41,  355;  Carpenter-Dallinger,  p.  288;  Nageli  and  Schwendener, 
p.  176;  Ranvier,  p.  29;  Robin,  p.  126;  Amer.  Soc.  Micrs.,  1884,  p. 
183;  1889,  p.  22;  Amer.  Jour.  Arts  and  Sciences,  1890,  p.  50;  Jour. 
Roy.  Micr.  Soc.,  1888,  1889;  1904,  pp.  261,  279;  A.  E.  Wright, 
Practical  Microscopy,  pp.  129,  145,  163. 

§  179.     Table  of  Magnification   and  of  the  Valuations  of 


126 


MAGNIFICATION  AND  MICROMETRY 


[_CH.  IV 


the  Ocular  Micrometer. —  The  table  should  be  filled  out  by  each 
student.  In  using  it  for  Micromeiry  and  Drawing  it  is  necessary  to 
keep  clearly  in  mind  the  exact  conditions  under  which  the  determinations 
were  made,  and  also  the  ways  in  which  variations  in  magnification  and 
the  variation  of  the  ocular  micrometer  may  be  produced  (§  177,  178, 
188,  195- 


OCULAR                                   OCULAR 

37  or  50  mm.                                     25  mm 

OBJECTIVE. 

TUBE 

IN 

TUBE 

OUT 
MM. 

TUBE 

IN 

TUBE 

OUT 
MM. 

OCULAR  MICROMETER 
VALUATION. 
TUBE  IN.     OUT  MM. 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

SIMPLE  MICROSCOPE.           X 

FIG.  115 
MICROMETRY 

§  180.      Micrometry  is  the  determination  of  the  size  of  objects 
by  the  aid  of  a  microscope. 

MICROMETRY    WITH    THE   SIMPLE    MICROSCOPE 


§  181.     With  a  simple  microscope   (A),   the  easiest  and  best 
way  is  to  use  dividers  and  then   with  the  simple  microscope  deter- 


("//.  IV}  MAGNIFICATION  AND  MICROMETRY  127 

mine  when  the  points  of  the  dividers  exactly  include  the  object. 
The  spread  of  the  dividers  is  then  obtained  as  above  (§  173).  This 
amount  will  be  the  actual  size  of  the  object,  as  the  microscope  was 
only  used  in  helping  to  see  when  the  divider  points  exactly  enclosed 
the  object,  and  then  for  reading  the  divisions  on  the  rule  in  getting 
the  spread  of  the  dividers. 

(B)  One  may  put  the  object  under  the  simple  microscope  and 
then,  as  in  determining  the  power  (§  172),  measure  the  image  at 
the  standard  distance.  If  the  size  of  the  image  so  measured  is 
divided  by  the  magnification  of  the  simple  microscope,  the  quotient 
give  the  actual  size  of  the  object.  One  might  use  the  eikonometer 
also  (§  196). 

Use  a  fly's  wing  or  some  other  object  of  about  that  size,  and 
try  to  determine  the  width  in  the  two  ways  described  above.  If  all 
the  work  is  accurately  done  the  results  will  agree. 

MICROMETRY    WITH    THE    COMPOUND    MICROSCPE 

There  are  several  ways  of  varying  excellence  for  obtaining  the 
size  of  objects  with  the  compound  microscope,  the  method  with  the 
ocular  micrometer  (§  189-193)  being  most  accurate. 

>J  182.  Unit  of  Measure  in  Micrometry.  —  As  most  of  the 
objects  measured  with  the  compound  microscope  are  smaller  than 
any  of  the  originally  named  divisions  of  the  meter,  and  the  common 
or  decimal  fractions  necessary  to  express  the  size  are  liable  to  be 
unnecessarily  cumbersome,  Harting,  in  his  work  on  the  microscope 
(1859),  proposed  the  one  thousandth  of  a  millimeter  (TTnnr  mm- 
or  o.ooi  mm.)  or  one  millionth  of  a  meter  (rinmnFTF  °r  o.oooooi 
meter)  as  the  unit.  He  named  this  unit  micro-millimeter  and 
designated  it  mmm.  In  1869,  Listing  (Carl's  Repetorium  fur  Ex- 
perimentai-Physik,  Bd,  X,  p.  5)  favored  the  thousandth  of  a  milli- 
meter as  unit  and  introduced  the  name  Mikron  or  micrum.  In 
English  it  is  most  often  written  Micron  (plural  micro,  or  microns, 
pronunciation  Mik'r6n  or  MIk'rSn).  By  universal  consent  the  sign 
or  abbreviation  used  to  designate  it  is  the  Greek  yu.  Adopting  this 
unit  and  sign,  one  would  express  five  thousandths  of  a  millimeter 
r  °-o°5  mm.)  thus,  5^.* 


*  The  term  micromillimeter,   abbreviation   mmm.,  is   very  cumbersome, 
and  besides  is  entirely  inappropriate  since  the  adoption  of  the  definite  mean- 


128  MAGNIFICATION  AND  MICROMETRY          [CH.1T 

'i  183.  Micrometry  by  the  use  of  a  stage  micrometer  on  which  to  mount 
the  object. — In  this  method  the  object  is  mounted  on  a  micrometer  and  then 
put  under  the  microscope,  and  the  number  of  spaces  covered  by  the  object  is 
read  off  directly.  It  is  exactly  like  putting  any  large  object  on  a  rule  and 
seeing  how  many  spaces  of  the  rule  it  covers.  The  defect  in  the  method  is 
that  it  is  impossible  to  properly  arrange  objects  on  the  micrometer.  Unless 
the  objects  are  circular  in  outline  they  are  liable  to  be  oblique  in  position,  and 
in  every  case  the  end  or  edges  of  the  object  may  be  in  the  middle  of  a  space 
instead  of  against  one  of  the  lines,  consequently  the  size  must  be  estimated  or 
guessed  at  rather  than  really  measured. 

§  184.  Micrometry  by  dividing  the  size  of  the  image  by  the 
magnification  of  the  microscope. — For  example,  employ  the  3  mm. 
(l/%  in.)  objective,  25  mm.  (i  in.)  ocular,  and  a  Necturus'  red  blood- 
corpuscle  preparation  as  object.  Obtain  the  size  of  the  image  of  the 
long  and  short  axes  of  three  corpuscles  with  the  camera  lucida  and 
dividers,  exactly  as  in  obtaining  the  magnification  of  the  microscope 
(§  176).  Divide  the  size  of  the  image  in  each  case  by  the  magnifi- 
cation, and  the  result  gives  the  actual  size  of  the  blood-corpuscles. 
Thus,  suppose  the  image  of  the  long  axis  of  the  corpuscle  is  18  mm. 
and  the  magnification  of  the  microscope  400  diameters  (§  170),  then 
the  actual  length  of  this  long  axis  of  the  corpuscle  is  18  mm.  -f-  400 
=0.045  mm-  or  45/^  (§  182). 


FIG.  116.  Preparation  of  blood  with 
a  ring  around  a  group  of  blood  cor- 
puscles. 


As  the  same  three  blood- corpuscles  are  to  be  measured  in  three 
ways,  it  is  an  advantage  to  put  a  delicate  ring  around  a  group  of 
three  or  more  corpuscles,  and  make  a  sketch  of  the  whole  enclosed 
group,  marking  on  the  sketch  the  corpuscles  measured  (Figs.  70, 
75).  The  different  corpuscles  vary  considerably  in  size,  so  that 
accurate  comparison  of  different  methods  of  measurement  can  only 


ings  for  the  prefixes  micro  and  mega,  meaning  respectively  one-millionth  and 
one  million  times  the  unit  before  which  it  is  placed.  A  micromillimeter 
would  then  mean  one-millionth  of  a  millimeter,  not  one-thousandth.  The 
term  micron  has  been  adopted  by  the  great  microscopical  societies,  the  inter- 
national commission  on  weights  and  measures,  and  by  original  investigators, 
and  is,  in  the  opinion  of  the  writer,  the  best  term  to  employ.  Jour.  Roy. 
Micr.  Soc.,  1888,  p.  502  ;  Nature,  Vol.  XXXVII  (1888),  p.  388. 


CH.  IV\          MAGNIFICATION  AND  MICROMETRY  129 

be  made  when  the  same  corpuscles  are    measured    in    each    of   the 
ways. 

§  185.  Micrometry  by  the  use  of  a  Stage  Micrometer  and  a 
Camera  Lucida. — Employ  the  same  object,  objective  and  ocular  as 
before.  Put  the  camera  lucida  in  position,  and  with  a  lead  pencil 
make  dots  on  the  paper  at  the  limits  of  the  image  of  the  blood- 
corpuscles.  Measure  the  same  three  that  were  measured  in  §  184. 

Remove  the  object,  place  the  stage  micrometer  under  the 
microscope,  focus  well,  and  draw  the  lines  of  the  stage  micrometer 
so  as  to  include  the  dots  representing  the  limits  of  the  part  of  the 
image  to  be  measured.  As  the  value  of  the  spaces  on  the  stage 
t  micrometer  is  known,  the  size  of  the  object  is  determined  by  the 
number  of  spaces  of  the  micrometer  required  to  include  it. 

This  simply  enables  one  to  put  the  image  of  a  fine  rule  on  the 
image  of  a  microscopic  object.  It  is  theoretically  an  excellent 
method,  and  nearly  the  same  as  measuring  the  spread  of  the  dividers 
with  a  simple  microscope  (§  173,  197). 

OCULAR     MICROMETER 

§  186.  Ocular  Micrometer,  Eye-Piece  Micrometer. — 
This,  as  the  name  implies,  is  a  micrometer  to  be  used  with  the 
ocular.  It  is  a  micrometer  on  glass,  and  the  lines  are  sufficiently 
coarse  to  be  clearly  seen  by  the  ocular.  The  lines  should  be  equi- 
distant and  TV  or  T\T  mtn.  apart,  every  fifth  line  should  be  longer 
and  heavier  to  facilitate  counting.  If  the  micrometer  is  ruled  in 
squares  (net  micrometer}  it  will  be  very  convenient  for  many  pur- 
poses. 

The  ocular  micrometer  is  placed  in  the  ocular,  no  matter  what 
the  form  of  the  ocular  (z.  <?.,  whether  positive  or  negative)  at  the 
level  at  which  the  real  image  is  formed  by  the  objective,  and  the 
image  appears  to  be  immediately  upon  or  under  the  ocular  microme- 
ter, and  hence  the  number  of  spaces  on  the  ocular  micrometer 
required  to  measure  the  real  image  may  be  read  off  directly.  This, 
however,  is  measuring  the  size  of  the  real  image,  and  the  actual 
size  of  the  object  can  only  be  determined  by  determining  the  ratio 
between  the  size  of  the  real  image  and  the  object.  In  other  words, 
it  is  necessary  to  get  the  valuation  of  the  ocular  micrometer  in  terms 
of  a  stage  micrometer. 


130 


MAGNIFICATION  AND  MICROMETRY          [  CH.  IV 


§  187.  Valuation  of  the  Ocular  Micrometer. — This  is  the 
value  of  the  divisions  of  the  ocular  micrometer  for  the  purposes  of 
micrometry,  and  is  entirely  relative,  depending  on  the  magnifica- 
tion of  the  real  image  formed  by  the  objective,  consequently  it 
changes  with  every  change  in  the  magnification  of  the  real  image, 
and  must  be  especially  determined  for  every  optical  combination 
(i.  e. ,  objective  and  ocular),  and  for  every  change  in  the  length  of 
the  tube  of  the  microscope.  That  is,  it  is  necessary  to  determine 
the  ocular  micrometer  valuation  for  every  condition  modifying  the 
real  image  of  the  microscope  (§  177). 

Any  Huygenian  ocular  (Fig.  117)  may,  however,  be  used  as  a  micrometer 
ocular  by  placing  the  ocular  micrometer  at  the  level  of  the  ocular  diaphragm, 
where  the  real  image  is  formed.  If  there  is  a  slit  in  the  side  of  the  ocular,  » 
and  the  ocular  micrometer  is  mounted  in  some  way  it  may  be  introduced 
through  the  opening  at  the  side.  When  no  side  opening  exists  the  mounting 
of  the  eye-lens  may  be  unscrewed  and  the  ocular  micrometer,  if  on  a  cover- 
glass  can  be  laid  on  the  upper  side  of  the  ocular  diaphragm. 


FIG.  117.     Sectional  view  of  a  Huygenian  ocular. 

Axis.  Optic  axis  of  the  ocular.  D.  Diaphragm  of 
the  ocular.  E.  L.  Eye-Lens.  F.  L.  Field-Lens. 

E.  P.  Eye-point.  In  micrometry  the  ocular  microm- 
eter with  a  Huygenian  ocular  must  be  placed  at  the  level 
of  the  diaphragm  where  the  real  image  is  formed.  In  a 
positive  ocular  it  would  be  placed  below  the  ocular  lenses. 


§  188.  Obtaining  the  Ocular  Micrometer  Valuation  for 
an  Ocular  Micrometer  with  fixed  Lines.— Use  the  stage 
micrometer  as  object.  Light  the  field  well  and  look  into  the  micro- 
scope. The  lines  of  the  ocular  micrometer  should  be  very  sharply 
defined.  If  they  are  not,  raise  or  lower  the  eye-lens  to  make  them 
so;  that  is,  focus  as  with  the  simple  magnifier. 

When  the  lines  of  the  ocular  micrometer  are  distinct,  focus  the 
microscope  (§  81,  84)  for  the  stage  micrometer.  The  image  of 
the  stage  micrometer  appears  to  be  directly  under  or  upon  the  ocular 
micrometer. 

Make  the  lines  of  the  two  micrometers  parallel  by  rotating  the 


€11.  IV\          MAGNIFICATION  AND  MICROMETRY  131 

ocular  or  changing  the  position  of  the  stage  micrometer  or  both  if 
necessary,  and  then  make  any  two  lines  of  the  stage  micrometer 
coincide  with  any  two  on  the  ocular  micrometer.  To  do  this  it  may 
be  necessary  to  pull  out  the  draw-tube  a  greater  or  less  distance. 
See  how  many  spaces  are  included  in  each  of  the  micrometers. 

Divide  the  value  of  the  included  space  or  spaces-  on  the  stage 
micrometer  by  the  number  of  divisions  on  the  ocular  micrometer 
required  to  include  them,  and  the  quotient  so  obtained  will  give  the 
valuation  of  the  ocular  micrometer  in  fractions  of  the  unit  of 
measure  of  the  stage  micrometer.  For  example,  suppose  the  milli- 
meter is  taken  as  the  unit  for  the  stage  micrometer  and  this  unit  is 
divided  into  spaces  of  T*T  and  T-^  millimeters.  If  with  a  given  optical 
combination  and  tube-length  it  requires  10  spaces  on  the  ocular  mi- 
crometer to  include  the  real  image  of  y1^  millimeter  on  the  stage  mi- 
crometer, obviously  one  space  on  the  ocular  micrometer  includes 
only  one-tenth  as  much, or  Y1^  mm.-;- io=T^  mm.  That  is,  each  space 
on  the  ocular  micrometer  includes  T^¥  of  a  millimeter  on  the  stage 
micrometer,  or  TjT  millimeter  of  the  length  of  any  object  under  the 
microscope,  the  conditions  remaining  the  same.  Or,  in  other  words, 
it  requires  100  spaces  on  the  ocular  micrometer  to  include  i  milli- 
meter on  the  stage  micrometer,  then  as  before,  i  space  of  the  ocular 
micrometer  would  have  a  valuation  of  T^  millimeter  for  the  pur- 
poses of  micrometry.  The  size  of  any  minute  object  may  be  deter- 
mined by  multiplying  this  valuation  of  one  space  by  the  number  of 
spaces  required  to  include  it.  For  example,  suppose  the  fly's  wing 
or  some  part  of  it  covered  8  spaces  on  the  ocular  micrometer,  it 
would  be  known  that  the  real  size  of  the  part  measured  is  y^j-  mm. 
X8— Tfjjmm.  or  80  /<  (§  182).  See  Mark,  Jour.  Applied  Micro- 
scopy, Vol.  I,  p.  4. 

§  189.  Micrometry  with  the  Ocular  Micrometer. — Use  the 
3  mm.  (^8  in.)  objective  with  the  preparation  of  Necturus  blood- 
corpuscles  as  object.  Make  certain  that  the  tube  of  the  microscope 
is  of  the  same  length  as  when  determining  the  ocular  micrometer 
valuation.  In  a  word,  be  sure  that  all  the  conditions  are  exactly  as 
when  the  valuation  was  determined,  then  put  the  preparation  under 
the  microscope  and  find  the  same  three  red  corpuscles  that  were 
measured  in  the  other  ways  (§  184-185). 

Count  the  divisions  on  the  ocular  micrometer  required  to  enclose 
or  measure  the  long  and  the  short  axis  of  each  of  the  three  cor- 


1 32  MAGNIFICATION  AND  MICROMETRY  [CH.  IV 

puscles,  multiply  the  number  of  spaces  in  both  cases  by  the  valuation 
of  the  ocular  micrometer  for  this  objective,  tube-length  and  ocular, 
and  the  results  will  represent  the  actual  length  of  the  axes  of  the 
corpuscles  in  each  case. 

The  same  corpuscle  is,  of  course,  of  the  same  actual  size,  when 
measured  in  each  of  the  three  ways,  so  that  if  the  methods  are  cor- 
rect and  the  work  carefully  enough  done,  the  same  results  should 
be  obtained  by  each  method.  (§  197.)* 


*  There  are  three  ways  of  using  the  ocular  micrometer,  or  of  arriving  at 
the  size  of  the  objects  measured  with  it : 

(A)  By  finding  the  value  of  a  division  of  the  ocular  micrometer  for  each 
optical  combination  and  tube-length  used,  and  employing  this  valuation  as  a 
multiplier.     This  is  the  method  given  in  the  text,  and  the  one  most  frequently 
employed.     Thus,  suppose  with  a  given  optical  combination  and  tube-length 
it  required  five  divisions  on  the  ocular  micrometer  to  include  the  image  of  j-,, 
millimeter  of  the  stage  micrometer,  then  obviously  one   space  on   the  ocular 
micrometer  would  include   \  of  fg  mm.  or  3V  mm.;  the  size  of  any  unknown 
object  under  the  microscope  would  be  obtained  by  multiplying  the  number  of 
divisions  on  the  ocular  micrometer  required  to  include  its  image  by  the  va.lue 
of  one  space,  or  in  this  case,  ^  mm.     Suppose  some  object,  as  the  fly's  wing, 
required  15  spaces  of  the  ocular  micrometer  to  include  some  part  of  it,  then 
the  actual  size  of  this  part  of  the  wing  would  be  i5XuV:="s>  or  °-6  mm. 

(B)  By  finding  the  number  of  divisions  on  the  ocular  micrometer  re- 
quired to  include  the  image  of  an  entire  millimeter  of  the  stage  micrometer, 
and  using  this  number  as  a  divisor.     This  number  is  also  sometimes  called  the 
ocular  micrometer  ratio.     Taking  the  same  case  as  in  (A),  suppose  five  divi- 
sions of  the  ocular  micrometer  are  required  to  include  the  image  of  ,%  mm., 
on  the  stage  micrometer,  then  evidently  it  would  require  5-=-  ^=25  divisions 
on  the  ocular  micrometer  to  include  a  whole  millimeter  on  the  stage  microme- 
ter, and  the  number  of  divisions  of  the  ocular  micrometer  required  to  measure 
an  object  divided  by  25  would  give  the  actual  size  of  the  object  in  millimeters 
or  in  a  fraction  of  a  millimeter.     Thus,  suppose  it  required  15  divisions  of  the 
ocular  micrometer  to  include  the  image  of  some  part  of  the   fly's  wing,  the 
actual  size  of  the  part  included  would  be  15-^-25=  f  or  0.6  mm.     This  method 
is  really  exactly  like  the  one  in  (A),  for  dividing  by  25  is  the  same  as  multi- 
plying by  &. 

(C)  By  having  the  ocular  micrometer  ruled  in  millimeters  and  divisions 
of  a  millimeter,  and  then  getting  the  size  of  the  real  image  in  millimeters. 
In  employing  this  method  a  stage  micrometer  is  used  as  object  and  the  size  of 
the  image  of  one  or  more  divisions  is  measured  by  the  ocular  micrometer, 
thus :  Suppose  the  stage  micrometer  is  ruled  TV  and  T^7  mm.  and  the  ocular 
micrometer  is  ruled  in  millimeters  and  r\y  mm.     Taking  T2<j  mm.  on   the  stage 
micrometer  as  object,  as  in  the  other  cases,  suppose  it  requires   10  of  the  ^ 
mm.    spaces   or   i    mm.    to   measure   the   real   image,  then   the    real    image 


CH. 


.//.  {UNIFICATION  AND  MICROMETRY 


133 


§  190.  Obtaining  the  Valuation  of  the  Filar  Micrometer.— 
This  micrometer  (Figs.  118-120)  usually  consists  of  a  Ramsden's 
ocular  and  cross  lines.  As  seen  in  Fig.  119  A  there  are  three  lines. 
The  horizontal  and  one  vertical  line  are  fixed.  One  vertical  line 
may  be  moved  by  the  screw  back  and  forth  across  the  field. 

For  obtaining  the  valuation  of  this  ocular  micrometer  an  ac- 

FIG.  1 1 8.  Ocular  Screw- Micrometer 
wit/i  compensation  ocular X  6.  The  upper 
figure  shows  a  sectional  view  of  the  ocular 
and  the  screw  for  moving  the  micrometer 
at  the  right.  At  the  left  is  shown  a 
damping  screw  to  fasten  the  ocular  to  the 
upper  part  of  the  microscope  tube.  Below 
is  a  face  viezv,  showing  the  graduation  on 
the  wheel.  An  ocular  micrometer  like  this 
is  in  general  like  the  cob-web  micrometer 
and  may  be  used  for  measuring  objects  of 
varying  sizes  very  accurately.  With  the 
ordinary  ocular  micrometer  very  small 
objects  frequently  fill  but  a  part  of  an  inter- 
val of  the  micrometer,  but  with  this  the 
movable  cross  lines  traverse  the  object  (or 
rather  its  real  image}  regardless  of  the  mi- 
nuteness of  the  object.  (Zeiss'  Catalog.) 


must  be  magnified  };;-h-T20-=5  diameters,  that  is,  the  real  image  is  five 
times  as  great  in  length  as  the  object,  and  the  size  of  an  object  may  be  deter- 
mined by  putting  it  under  the  microscope  and  getting  the  size  of  the  real 
image  in  millimeters  with  the  ocular  micrometer  and  dividing  it  by  the  mag- 
nification of  the  real  image,  which  in  this  case  is  5  diameters. 

Use  the  fly's  wing  as  object,  as  in  the  other  cases,  and  measure  the  image 
of  the  same  part.  Suppose  that  it  required  30  of  the  ^  mm.  divisions  = 
, '•"  mm.  or  3  mm.  to  include  the  image  of  the  part  measured,  then  evidently 
the  actual  size  of  the  part  measured  is  3  mm.  -4-5=' |  mm.,  the  same  result  as 
in  the  other  cases.  See  also  \  195-196  on  the  Eikonometer. 

In  comparing  these  methods  it  will  be  seen  that  in  the  first  two  (A  andB) 
the  ocular  micrometer  may  be  simply  ruled  with  equidistant  lines  without 
regard  to  the  absolute  size  in  millimeters  or  inches  of  the  spaces.  In  the  last 
method  the  ocular  micrometer  must  have  its  spaces  some  known  division  of  a 
millimeter  or  inch.  In  the  first  two  methods  only  one  standard  of  measure  is 
required,  viz.:  the  stage  micrometer;  in  the  last  method  two  standards  must 
be  used, — a  stage  micrometer  and  an  ocular  micrometer.  Of  course,  the  ocular 
micrometer  in  the  first  two  cases  must  have  the  lines  equidistant  as  well  as  in 
the  last  case,  but  ruling  lines  equidistant  is  quite  a  different  matter  from  get- 
ting them  an  exact  division  of  a  millimeter  or  of  an  inch  apart. 


134 


MA  GNIFICA  TION  AND  MICR  OME  TR  Y          [  CH.  IV 


curate  stage  micrometer  must  be  used.  Carefully  focus  the  y^ 
mm.  spaces.  The  lines  of  the  ocular  micrometer  should  also  be 
sharp.  If  they  are  not  focus  them  by  moving  the  top  of  the  ocular 
up  or  down  (§  188).  Make  the  vertical  lines  of  the  filar  micrometer 
parallel  with  the  lines  of  the  stage  micrometer.  Take  the  precau- 
tions regarding  the  width  of  the  stage  micrometer  lines  given  in 
§  197  (see  also  Fig.  123).  Note  the  position  of  the  graduated 
wheel  and  of  the  teeth  of  the  recording  comb,  and  then  rotate  the 
wheel  until  the  movable  line  traverses  one  space  on  the  stage  mi- 
crometer. Each  tooth  of  the  recording  comb  indicates  a  total 
revolution  of  the  wheel,  and  by  noting  the  number  of  teeth  required 
and  the  graduations  on  the  wheel,  the  revolutions  and  part  of  a 
revolution  required  to  measure  the  y-^  mm.  of  the  stage  micrometer 


FIG.  119.  Filar  Micrometer  Ocular.  This  filar  micrometer  ocular  is  of 
the  Ramsden  type  and  consists  of  a  positive  ocular  with  a  moveable  hair  line 
and  two  reference  lines  at  right  angles  to  each  other  as  shown  in  A.  The 
moveable  line  must  be  carried  over  the  entire  length  of  the  object  to  be  meas- 
ured by  rotating  the  drum. 

A.  Field  of  the  filar  micrometer  showing  the  moveable  and  the  cross 
lines,  and  the  comb.  The  teeth  serve  to  measure  the  total  revolutions  of  the 
drum.  (Cut  loaned  by  the  Bausch  &  Lomb  Optical  Co. ) 

can  be  easily  noted.  Measure  in  like  manner  4  or  5  spaces  and  get 
the  average.  Suppose  this  average  is  i^  revolutions  or  125  grad- 
uations on  the  wheel,  to  measure  the  T|¥  mm.  or  io//  (see  §  182), 
then  one  of  the  graduations  on  the  wheel  would  measure  10/1  divided 
by  125— .o8/*.  In  using  this  valuation  for  actual  measurement,  the 
tube  of  the  microscope  and  the  objective  must  be  exactly  as  when 
obtaining  the  valuation  (see  §  187,  194). 


Cll.  //']          MAGNIFICATION  AND  MICROMETRY  135 

£  191.  Example  of  Measurement. — Suppose  one  uses  the 
red  blood  corpuscles  of  a  dog  or  monkey,  etc.,  every  condition  being 
as  when  the  valuation  was  determined,  one  notes  very  accurately 
how  many  of  the  graduations  on  the  wheel  are  required  to  make  the 
movable  line  traverse  the  object  from  edge  to  edge.  Suppose  it 
requires  94  of  the  graduations  to  measure  the  diameter,  the  actual 
size  of  the  corpuscle  would  be  94  X  .o8//~7-52yu. 

The  advantage  of  the  filar  micrometer  is  that  the  valuation  of 
one  graduation  being  so  small,  even  the  smallest  object  to  be  meas- 
ured would  require  several  graduations  to  measure  it.  In  ocular 
micrometers  with  fixed  lines,  small  objects  like  bacteria  might  not 
fill  even  one  space,  therefore  estimations,  not  measurements,  must 
be  made.  For  large  objects,  like  most  of  the  tissue  elements,  the 
ocular  micrometers  with  fixed  lines  answer  very  well,  for  the  part 
which  must  be  estimated  is  relatively  small  and  the  chance  of  error 
is  correspondingly  small. 

§  192.  Obtaining  the  Valuation  of  the  Combined  Ocular 
Micrometer  (Fig.  120). — To  obtain  the  valuation  of  this  ocular 
micrometer  one  proceeds  exactly  as  for  the  micrometer  with  fixed 
lines  (§  1 88),  except  that  a  partial  stage  micrometer  space  can  be 
measured  by  rotating  the  drum  until  the  ocular  micrometer  exactly 
coincides  with  the  stage  micrometer.  One  can  then  count  up  the 
number  of  spaces  on  the  ocular  micrometer  required  to  measure 
one  or  more  spaces  on  the  stage  micrometer.  To  this  is  then 
added  the  100  hundredths  of  a  space  indicated  on  the  drum.  For 
example  suppose  that  it  required  7  complete  spaces  of  the  ocular 
micrometer  and  the  drum  showed  50  hundredths  to  measure  3 
spaces  (3  hundredths  mm.)  on  the  stage  micrometer,  then  each 
space  on  the  ocular  micrometer  would  be  equal  to  0.03  mm. -5-7.50= 
0.004  mtn-  or  4/*-  One  °f  tne  spaces  on  the  drum  which  represents 
one  hundredth  of  an  interval  on  the  ocular  micrometer  would  have 
a  valuation  under  these  conditions  of  only  0.04/4.  This  gives  a 
clear  notion  of  the  minuteness  of  the  objects  which  can  be  measured 
and  of  the  smallness  of  the  error  in  measuring  large  objects  even  if 
one  should  get  the  object  a  few  of  the  drum  divisions  too  small  or 
too  large. 

§  193.  Example  of  Measurement  with  the  Combined 
Ocular  Micrometer. — Select  an  oval  corpuscle  of  some  lower 


136 


MAGNIFICATION  AND  MICROMETRY          [CH.  IV 


animal  (frog,  hen,  turtle,  etc.).  Arrange  the  micrometer  ocular  so 
that  the  long  axis  of  the  corpuscle  will  coincide  with  the  cross  line 
in  the  micrometer  scale  (Fig.  121).  Get  one  end  of  the  corpuscle 
exactly  level  with  one  end  of  the  micrometer  scale.  Note  the  posi- 
tion of  the  drum,  and  then  rotate  it  until  the  other  end  of  the 
corpuscle  is  exactly  against  the  nearest  line  of  the  micrometer. 
Count  up  the  entire  intervals  required  and  the  partial  interval  on 
the  drum.  Suppose  it  requires  5  entire  and  0.60  intervals  (see 
explanation  of  Fig.  121)  then  the  whole  corpuscle  must  be  5.60 
intervals  multiplied  by  4^.  the  value  of  one  interval:  5.6X4= 

21. 

FIG.  120.  Screw  Ocular 
Micrometer  with  moveable 
scale.  This  is  a  Huygenian 
ocular  zvith  a  5  nun.  scale 
divided  into  20  %  nun.  in- 
tervals. The  pitch  of  the 
screw  moving  the  scale  is 


14  mm.  therefore  one  com- 
plete revolution  of  the  drum 
moves  the  scale  one  interval 
or  ^4  mm.  The  drum  is 
divided  into  100  gradua- 
tions thus  enabling  one  to 
measure  woth  of  an  interval 
on  the  micrometer  scale. 
This  ocular  micrometer  combines  the  advantages  of  the  ocular  micrometer 
with  fixed  scale  and  the  filar  micrometer.  To  complete  the  measurement  of 
an  object  not  exactly  between  any  two  of  the  micrometer  lines  the  drum  need 
be  revolved  only  partly  around.  (Cut  loaned  by  the  Spencer  Lens  Co.  ) 

o      12345  FIG.  121.     Figure  of  the  scale  of  the  screw  ocular 

micrometer,  showing  the  divisions  and  the  cross  line. 
At  the  left  is  shown  an  object  on  the  scale  not  quite  fill- 
ing fo  of  the  intervals.  To  measure  this  the  drum  need 
be  revolved  only  sufficiantly  to  measure  the  part  of  the  interval  filled  by  the 
object  being  measured. 

'Originally  the  scale  was  divided  in  50  ^  mm.  spaces,  and  no  cross  line 
was  present.     In  7905  the  present  form  of  scale  was  specially  prepared  from 
the  writer's  specifications,  and  has  since  that  time  been  regularly  supplied. 
(Cut  loaned  by  the  Spencer  Lens  Co.) 

§  194.  Varying  the  Ocular  Micrometer  Valuation.  —  Any 
change  in  the  objective,  the  ocular  or  the  tube-length  of  the  micro- 
scope, that  is  to  say,  any  change  in  the  size  of  the  real  image,  pro- 


CH.  I V  ]  MA  GNIFICA  TION  AND  MICRO  ME  TRY  137 

duces  a  corresponding  change   in   the  ocular  micrometer  valuation 

(S  177,  187,  197). 

§  195.  Eikonometer  for  Magnification  and  Micrometry.— 
The  eikonometer  is  something  like  an  eye.  It  has  a  converging 
lens  serving  in  place  of  the  crystalline  lens  to  focus  the  rays  from 
the  eye-piece  of  the  compound  microscope,  or  from  the  simple  micro- 
scope upon  a  micrometer  scale,  the  scale  taking  the  place  of  the 
retina  in  the  eye  (Fig.  16).  This  scale  is  ruled  in  T:T  tnms.  Above 
the  scale  is  a  Ramsden's  ocular  of  25  mm.  equivalent  focus,  giving 
a  magnification  of  10.  The  eikonometer  scale  therefore  is  a  milli- 
meter scale  when  seen  at  the  distance  of  250  mm.  in  the  visual  field 
of  the  normal  human  eye,  and  it  enables  one  to  put  a  millimeter 
scale  on  the  image  of  any  object  studied. 

To  use  it  for  magnification  a  stage  micrometer  is  put  under  the 
microscope  and  carefully  focused.  Then  the  eikonometer  is  put  in 
place  over  the  ocular.  The  microscopic  image  of  the  stage  microm- 
eter and  the  scale  of  the  eikonometer  will  then  appear  in  the  same 
field  as  with  the  ordinary  ocular  micrometer  (§  188).  The  two  sets 
of  lines  should  be  made  parallel.  See  how  many  divisions  of  the 
eikonometer  millimeter  scale  are  required  to  measure  one  or  more 
of  the  divisions  of  the  image  of  the  stage  micrometer.  Suppose  it 
requires  6  intervals  or  millimeters  of  the  eikonometer  scale  to  meas- 
ure the  image  of  T§T)-  mm.  on  the  stage  micrometer.  The  size  of  the 
object  is  then  T-§- ,  mm.  and  of  its  image  6  mm.  The  magnification 
is  therefore  (§  170)  6  nim.-j-Tfir=2cx). 

For  determining  the  magnification  of  a  simple  microscope  the 
eikonometer  is  placed  over  the  simple  microscope  as  it  was  over  the 
ocular  above.  With  this  instrument  as  with  the  camera  lucida  only 
one  eye  is  used  (§  176). 

§  196.  Micrometry  with  the  Eikonometer. — In  the  first 
place  the  magnification  of  the  microscope  must  be  determined 
as  described  in  the  preceding  section  ;  and  one  must  keep  in  mind 
the  factors  which  will  vary  the  magnification  (§  177).  The  object 
to  be  measured  is  put  under  the  microscope  and  focused  and  the 
eikonometer  put  in  position.  The  virtual  image  is  then  measured  in 
millimeters  by  the  eikonometer  mm.  scale.  The  size  of  this  virtual 
image  is  then  divided  by  the  magnification  and  the  result  will  be 
the  actual  size  of  the  object  as  in  (§  184). 


138  MAGNIFICATION  AND  MICROMETRY          [CH.  IV 

For  example  suppose  the  long  axis  of  a  Necturus'  red  blood 
corpuscle  measures  9  mm.  on  the  eikonometer  scale.  If  the  magni- 
fication of  the  microscope  is  200  as  found  above  then  the  actual 
length  of  the  corpuscle  is  9  mm. -1-200= 0.045  mm.,  or  45/1.  (See 
A.  E.  Wright,  Jour.  Roy.  Micr.  Soc.,  1904,  pp.  261,  279;  Princi- 
ples of  Microscopy,  pp.  145,  163.) 


D 

>!  x    ^  ^ 

£"  MICROSCOPE  EIKOI.OMETER 

FIG.  122.       Wright's  Eikonometer  for  Magnification  and  Micrometry  — 
(from  Beck's  Catalog.) 

A.  Objective;  B.  Ocular;  D.  The  object;  E.  Virtual  image  of  the 
microscope ;  C.  The  Eikonometer  placed  over  the  ocular.  The  lens  G, 
produces  a  real  image  on  the  eikonometer  scale  at  F.  This  scale  and  real 
image  are  then  viewed  through  the  Ramsden  ocular  of  2j  mm.  equivalent 
focus,  H. 

\  197.  Remarks  on  Micrometry. — In  using  adjustable  objectives  ($  27, 
114),  the  magnification  of  the  objective  varies  with  the  position  of  the  adjust- 
ing collar,  being  greater  when  the  adjustment  is  closed  as  for  thick  cover- 
glasses  than  when  open,  as  for  thin  ones.  This  variation  in  the  magnification 
of  the  objective  produces  a  corresponding  change  in  the  magnification  of  the 
entire  microscope,  and  the  ocular  micrometer  valuation — therefore  it  is  neces- 
sary to  determine  the  magnification  and  ocular  micrometer  valuation  for  each 
position  of  the  adjusting  collar. 

While  the  principles  of  micrometry  are  simple,  it  is  very  difficult  to  get 
the  exact  size  of  microscopic  objects  This  is  due  to  the  lack  of  perfection 
and,  uniformity  of  micrometers,  and  the  difficulty  of  determining  the  exact 
limits  of  the  object  to  be  measured.  Hence,  all  microscopic  measurements 
are  only  approximately  correct,  the  error  lessening  with  the  increasing  perfec- 
tion of  the  apparatus  and  the  skill  of  the  observer. 

A  difficulty  when  one  is  using  high  powers  is  the  width  of  the  lines  of 
the  micrometer.  If  the  micrometer  is  perfectly  accurate  half  the  width  of 
each  line  belongs  to  the  contiguous  spaces,  hence  one  should  measure  the 
image  of  the  space  from  the  centers  of  the  lines  bordering  the  space,  or  as 
this  is  somewhat  difficult  in  using  the  ocular  micrometer,  one  may  measure 


CH. 


MAGNIFICATION  AND  MICROMETRY 


139 


from  the  inside  of  one  bordering  line  and  from  the  outside  of  the  other.  If 
the  lines  are  of  equal  width  this  is  as  accurate  as  measuring  from  the  center  of 
the  lines.  Evidently  it  would  not  be  right  to  measure  from  either  the  inside 
or  the  outside  of  both  lines  (Fig.  123). 

It  is  also  necessary  in  micrometry  to  use  an  objective  of  sufficient  power 
to  enable  one  to  see  all  the  details  of  an  object  with  great  distinctness.  The 
necessity  of  using  sufficient  amplification  in  micrometry  has  been  es- 
pecially remarked  upon  by  Richardson,  Monthly  Micr.  Jour.,  1874,  1875,; 
Rogers,  Proc.  Amer.  Soc.  Microscopists,  1882,  p.  239;  Ewell,  North  American 
Pract.,  1890,  pp.  97,  173. 

FIG.  123.     The  appearance  of  the 

coarse  stage  micrometer  and  of  the  *  R 

fine  ocular   micrometer   lines    when 
using  a  high  objective. 

(A}.  The  method  of  measuring 
the  spaces  by  putting  the  fine  ocular 
micrometer  lines  opposite  the  center 
of  the  course  stage  micrometer  lines. 

(B).  Method  of  measuring  the 
spaces  of  the  stage  micrometer  6y 
one  line  of  the  ocular  micrometer 
(o.  in.)  at  the  inside  and  one  at  the 
outside  of  the  course  stage  microm- 
eter lines  (s.  /«.). 

As  to  the  limit  of  accuracy  in  micrometry,  one  who  has  justly  earned  the 
right  to  speak  with  authority  expresses  himself  as  follows:  "/  assume  that 
o.2/.i  is  the  limit  of  precision  in  microscopic  measures  beyond  which  it  is  im- 
possible to  go  with  certainty."  W.  A.  Rogers  Proc.  Amer.  Soc.  Micrs. ,  1883,  p. 
198. 

In  comparing  the  methods  of  micrometry  with  the  compound  microscope 
given  above  (#  183,  184,  185,  189,  191,  193,  196,),  the  one  given  in  §  183  is 
impracticable,  that  given  in  §  184  is  open  to  the  objection  that  two  standards 
are  required, — the  stage  micrometer,  and  the  steel  rule;  it  is  open  to  the  fur- 
ther objection  that  several  different  operations  are  necessary,  each  operation 
adding  to  the  probability  of  error.  Theoretically  the  method  given  in  \  185  is 
good,  but  it  is  open  to  the  very  serious  objection  in  practice  that  it  requires  so 
many  operations  which  are  especially  liable  to  introduce  errors.  The  method 
that  experience  has  found  most  safe  and  expeditious,  and  applicable  to  all 
objects,  is  the  method  with  the  ocular  micrometer.  If  the  valuation  of  the 
ocular  micrometer  has  been  accurately  determined,  then  the  only  difficulty  is 
in  deciding  on  the  exact  limits  of  the  objects  to  be  measured  and  so  arranging 
the  ocular  micrometer  that  these  limits  are  inclosed  by  some  divisions  of  the 
micrometer.  Where  the  object  is  not  exactly  included  by  whole  spaces  on  the 
ocular  micrometer,  the  chance  of  error  comes  in,  in  estimating  just  how  far 
into  a  space  the  object  reaches  on  the  side  not  in  contact  with  one  of  the  mi- 
crometer lines.  If  the  ocular  micrometer  has  some  quite  narrow  spaces,  and 


140  MAGNIFICATION  AND  MICROMETRY          \_CH.IV 

others  considerably  larger,  one  can  nearly  always  manage  to  exactly  include 
the  object  by  some  two  lines.  The  ocular  screw  micrometers  (Figs.  118-120) 
obviate  this  entirely  as  the  cross  hair  or  lines  traverse  the  object  or  its  real 
image,  and  whether  this  distance  be  great  or  small  it  can  be  read  off  on  the 
graduated  wheel,  and  no  estimation  or  guess  work  is  necessary. 

The  new  method  by  means  of  Wright's  Eikonometer  (\  \  195-6)  is  spoken 
of  very  favorably  by  experts  who  have  employed  it.  For  those  especially  in- 
terested in  micrometry,  as  in  its  relation  to  medical  jurisprudence,  the  follow- 
ing references  are  recommended.  These  articles  consider  the  problem  in  a 
scientific  as  well  as  a  practical  spirit:  The  papers  of  Prof.  \Vm.  A.  Rogers  on 
micrometers  and  micrometry,  in  the  Amer.  Quar.  Micr.  Jour.,  Vol.  I.  pp.  97, 
208;  Proceedings  Amer.  Soc.  Microscopists,  1882,  1883,  1887.  Dr.  M.  D.  Ewell, 
Proc.  Amer.  Soc.  Micrs. ,  1890;  The  Microscope,  1889,  pp.  43-45;  North  Amer. 
Pract.,  1890,  pp.  97,  173.  Dr.  J.  J.  Woodward,  Amer.  Jour,  of  the  Med.  Sci., 
1875.  M.  C.  White,  Article  "  Blood-stains,"  Ref.  Hand-book  Med.  Sciences, 
1885.  Medico-Legal  Journal,  Vol.  XII.  For  the  change  in  magnification  due 
to  a  change  in  the  adjustment  of  adjustable  objectives,  see  Jour.  Roy.  Micr. 
Soc.  1880,  p.  702;  Amer.  Monthly  Micr.  Jour.,  1880,  p.  67.  Carpenter-Dallinger, 
p.  270  and  end  of  \  196. 

If  one  consults  the  medico-legal  journals;  the  microscopical  journals,  the 
Index  Medicus,  and  the  Index  Catalog  of  the  Library  of  the  Surgeon  General's 
Office,  under  Micrometry,  Blood,  and  Jurisprudence,  he  can  get  on  track  of  the 
main  work  which  has  been  and  is  being  done. 


10     CENTIMETER     RULE 

The  upper  edge  is  in  millimeters,    the   lower   in   centimeters,  and   half 
centimeters. 

THE    METRIC    SYSTEM 


UNITS.  The  most  commonly  used  divisions  and  multiples 

,«.  »cc-n-Cr,  TT,-,T>  (   Centimeter  (c.  m. ) ,  i-iooth  Meter;    Millimeter   (m.m.),    i-ioooth    Meter: 

t  Vp  \  Micron  (/<  ),i-ioooth  Millimeter;  the  Micron  is  the  unit  in  Micrometry(§i66). 

'     '      (    Kilometer,  1000  Meters;  used  in  measuring  roads  and  other  long  distances. 

THE  GRAM  FOR    (   Milligram  (in.  g.),  i-ioooth  Gram. 
WEIGHT     .     .     \   Kilogram,  1000  Grams,  used  for  ordinary  masses,  like  groceries,  etc. 

THE  LITER  FOR  /    Cubic  Centimeter  (c.  c.),  i-ioooth  I,iter.      This  is  more  common  than  the 
CAPACITY    .       [  correct  form,  Milliliter. 

Divisions  of  the  L'nits  are  indicated  by  the  l,atin  prefixes;     deci,  i-ioth  ;  centi.  t-iooth  ; 
Milli,  i-ioooth  ;  Micro,  i-i,ooo,oooth  of  any  unit. 

Multiples  are  designated  by  Greek  prefixes  :    deka,  10  times      hecto,  100  times  ;    kilo,  1000 
times  ;  myria  10,000  times  ;  Mega,  1,000,000  times  any  unit. 


CHAPTER   V 


DRAWING  WITH  THE  MICROSCOPE 


APPARATUS    AND    MATERIAL    FOR    THIS    CHAPTER 

Microscope,  Abbe  and  Wollaston's  camera  lucidas,  drawing  board,  thumb 
tacks,  pencils,  paper,  and  microscope  screen,  (Fig.  66),  microscopic  prepara- 
tions. 

DRAWING    MICROSCOPIC    OBJECTS 

§  198.  Microscopic  objects  may  be  drawn  free-hand  directly 
from  the  microscope,  but  in  this  way  a  picture  giving  only  the  gen- 
eral appearance  and  relations  of  parts  is  obtained.  For  pictures 
which  shall  have  all  the  parts  of  the  object  in  true  proportions  and 
relations,  it  is  necessary  to  obtain  an  exact  outline  of  the  image  of 
the  object,  and  to  locate  in  this  outline  all  the  principal  details  of 
structure.  It  is  then  possible  to  complete  the  picture  free-hand 
from  the  appearance  of  the  object  under  the  microscope  The  ap- 
pliance used  in  obtaining  outlines,  etc.,  of  the  microscope  image  is 
known  as  a  camera  lurida. 

§  199.  Camera  Lucida. — This  is  an  optical  apparatus  for  en- 
abling one  to  see  objects  in  greatly  different  situations,  as  if  in  one 
field  of  yision,  and  with  the  same  eye.  In  other  words  it  is  an  opti- 
cal device  for  superimposing  or  combining  two  fields  of  view  in  one 
eye. 

As  applied  to  the  microscope,  it  causes  the  magnified  virtual 
image  of  the  object  under  the  microscope  to  appear  as  if  projected 
upon  the  table  or  drawing  board,  where  it  is  visible  with  the  draw- 
ing paper,  pencil,  dividers,  etc.,  by  the  same  eye,  and  in  the  same 
field  of  vision.  The  microscopic  image  appears  like  a  picture  on  the 
drawing  paper  (see  note  to  §  202).  This  is  accomplished  in  two 
distinct  ways: 

(A)  By  a  camera  lucida  reflecting  the  rays  from  the  microscope 
so  that  their  direction  when  they  reach  the  eye  coincides  with  that 


142 


DRA  WING  WITH  THE  MICROSCOPE 


\_CH.    V 


of  the  rays  from  the  drawing  paper,  pencil,  etc.  In  some  of  the 
camera  lucidas  from  this  group  (Wollaston's,  Figs.  108,  112),  the 
rays  are  reflected  twice,  and  the  image  appears  as  when  looking 


FIG.  125 


FIG.  126 


FIG.  124 


FIG.  124.  Abbe  Camera  Lucida 
with  the  mirror  at  45° ,  the  drawing 
surface  horizontal,  and  the  micro- 
scope vertical. 

Axis,  Axis.  Axial  ray  from  the 
microscope  and  from  the  drawing 
surface.  A,  B.  Marginal  rays  of 
the  field  on  the  drawing  surface,  a  b. 
Sectional  view  of  the  silvered  surface 

on  the  upper  of  the  triangular  prisms  composing  the  cubical  prism  (P).  The 
silvered  surface  is  shown  as  incomplete  in  the  center,  thus  giving  passage  to 
the  rays  from  the  microscope. 

Foot.     Foot  or  base  of  the  microscope. 

G.  Smoked  glass  seen  in  section.  It  is  placed  between  the  mirror  and  the 
prism  to  reduce  the  light  from  the  drawing  surface. 

Mirror.  The  mirror  of  the  camera  lucida.  A  Quadrant  (Q)  has  been 
added  to  indicate  the  angle  of  inclination  of  the  mirror,  which  in  this  case  is 

45°- 

1     Ocular.     Ocular  of  the  microscope  over  which  the  prism  of  the  camera 
lucida  is  placed. 

P,  P.-    Drawing  pencil  and  the  cubical  prism,  over  the  ocular. 

FIG.  125.  Geometrical  figure  showing  the  angles  made  by  the  axial  ray 
with  the  drawing  surface  aud  the  mirror. 

A,  B.     The  drawing  surface. 

FIG.  126.  Ocular  showing  eye-point,  E.  P.  It  is  at  this  point  both  hor- 
izontally and  vertically  that  the  hole  in  the  silvered  surface  should  be  placed 
($203). 


en.  v]          />A'.-ni7M;  \\~rrn  THE  MICROSCOPE  i43 

directly  into  the  microscope.  In  others  the  rays  are  reflected  but 
once,  and  the  image  has  the  inversion  produced  by  a  plane  mirror. 
For  drawing  purposes  this  inversion  is  a  great  objection,  as  it  is 
necessary  to  similarly  invert  all  the  details  added  free-hand. 

(B)  By  a  camera  lucida  reflecting  the  rays  of  light  from  the 
drawing  paper,  etc.,  so  that  their  direction  when  they  reach  the  eye 
coincides  with  the  direction  of  the  rays  from  the  microscope  (Fig. 
65,  124).  In  all  of  the  camera  lucidas  of  this  group,  the  rays  from 
the  paper  are  twice  reflected  and  no  inversion  appears. 

The  better  forms  of  camera  lucidas  (Wollaston's,  Grunow's, 
Abbe's,  etc.),  may  be  used  for  drawing  both  with  low  and  with 
high  powers.  Some  require  the  microscope  to  be  inclined  (Fig. 
in)  while  others  are  designed  to  be  used  on  the  microscope  in  a 
vertical  position.  As  in  biological  work,  it  is  often  necessary  to 
have  the  microscope  vertical,  the  form  for  a  vertical  microscope  is 
to  be  preferred  ;  but  see  Fig.  130. 

§  200.  Avoidance  of  Distortion. — In  order  that  the  picture 
drawn  by  the  aid  of  a  camera  lucida  may  not  be  distorted,  it  is  neces- 
sary that  the  axial  ray  from  the  image  on  the  drawing  surface  shall  be 
at  right  angles  to  the  drawing  surface  (Figs.  127,  129). 

£  201.  Wollaston's  Camera  Lucida. — This  is  a  quadrangular  prism  of 
glass  put  in  the  path  of  the  rays  from  the  microscope,  and  it  serves  to  change 
the  direction  of  the  axial  ray  90  degrees.  In  using  it  the  microscope  is  made 
horizontal,  and  the  rays  from  the  microscope  enter  one-half  of  the  pupil  while 
rays  from  the  drawing  surface  enter  the  other  half  of  the  pupil.  As  seen  in 
figure  127,  the  fields  partly  overlap,  and  where  they  do  so  overlap,  pen- 
cil or  dividers  and  microscopic  image  can  be  seen  together. 

In  drawing  or  using  the  dividers  with  the  Wollaston  camera  lucida  it  is 
necessary  to  have  the  field  of  the  microscope  and  the  drawing  surface  about 
equally  lighted.  If  the  drawing  surface  is  too  brilliantly  lighted  the  pencil  or 
dividers  may  be  seen  very  clearly,  but  the  microscopic  image  will  be  obscure. 
On  the  other  hand,  if  the  field  of  the  microscope  has  too  much  light  the 
microscopic  image  will  be  very  definite,  but  the  pencil  or  dividers  will  not  be 
visible.  It  is  necessary,  as  with  the  Abbe  camera  lucida  (\  203),  to  have  the 
Wollaston  prism  properly  arranged  with  reference  to  the  axis  of  the  micro- 
scope and  the  eye-point.  If  it  is  not,  one  will  be  unable  to  see  the  image  well, 
and  may  be  entirely  unable  to  see  the  pencil  and  the  image  at  the  same  time. 
Again,  as  rays  from  the  microscope  and  from  the  drawing  surface  must  enter 
independent  parts  of  the  pupil  of  the  same  eye,  one  must  hold  the  eye  so  that 
the  pupil  is  partly  over  the  camera  lucida  and  partly  over  the  drawing  surface. 
One  can  tell  the  proper  position  by  trial.  This  is  not  a  very  satisfactory 
camera  to  draw  with,  but  it  is  a  very  good  form  to  measure  the  vertical  dis- 


144 


DRAWING  WITH  THE  MICROSCOPE 


[CH.    V 


tance  of  250  mm.  at  which  the  drawing  surface  should  be  placed  when  deter- 
mining magnification  (\  178). 

§  202.  *Abbe  Camera  Lucida. — This  consists  of  a  cube  of 
glass  cut  into  two  triangular  prisms  and  silvered  on  the  cut  surface 
of  the  upper  one.  A  small  oval  hole  is  then  cut  out  of  the  center  of 
the  silvered  surface  and  the  two  prisms  are  cemented  together  in  the 
form  of  the  original  cube  with  a  perforated  45  degree  mirror  within 
it  (Fig.  124,  a  b).  The  upper  surface  of  the  cube  is  covered  by  a 
perforated  metal  plate.  This  cube  is  placed  over  the  ocular  in  such 
a  way  that  the  light  from  the  microscope  passes  through  the  hole  in 
the  silvered  face  and  thence  directly  to  the  eye.  Light  from  the 
drawing  surface  is  reflected  by  a  mirror  to  the  silvered  surface  of 
the  prism  and  reflected  by  this  surface  to  the  eye  in  company  with 
the  rays  from  the  microscope,  so  that  the  two  fields  appear  as  one, 
and  the  image  is  seen  as  if  on  the  drawing  surface  (Figs.  124,  129). 
It  is  designed  for  use  with  a  vertical  microscope.  [Compare  §  205.] 


FIG.  127.  Wollaston's  Cam- 
era Lucida,  showing  the  rays 
from  the  microscope  and  from  the 
drawing  surface,  and  the  position 
of  the  pupil  of  the  eye.  See  also 
Fig.  112. 

For  full  explanation  see  Fig.  108 


*For  some  persons  the  image  and  drawing  surface.pencil,  etc. ,  do  not  appear 
on  the  drawing  board  as  stated  above,  but  under  the  microscope,  according 
to  the  general  principle  that  "objects  appear  in  space  where  they  could  be 
touched  along  a  perpendicular  to  the  retinal  surface  stimulated," — that  is  in 
the  line  of  rays  entering  the  eye.  This  is  always  the  case  with  the  Wollaston 
camera  lucida.  The  explanation  of  the  apparent  location  of  the  image,  etc., 
on  the  drawing  board  with  the  Abbe  camera  lucida  is  that  the  attention  is  con- 
centrated upon  the  drawing  surface  rather  than  upon  the  object  under  the 
microscope  (Dr.  W.  B.  Pillsbury). 


c//.  /'] 


DRAWING  li'/T/l  THE  MICROSCOPE 


MS 


§  203.  Arrangement  of  the  Camera  Lucida  Prism. — In 
placing  this  camera  lucida  over  the  ocular  for  drawing  or  the  deter- 
mination of  magnification,  the  center  of  the  hole  in  the  silvered 
surface  is  placed  in  the  optic  axis  of  the  microscope.  This  is  done 
by  properly  arranging  the  centering  screws  that  clamp  the  camera 
to  the  microscope  tube  or  ocular.  The  perforation  in  the  silvered 
surface  must  also  be  at  the  level  of  the  eye-point.  In  other  words 
the  prism  must  be  so  arranged  vertically  and  horizontally  that  the 
hole  in  the  silvered  surface  is  in  the  axis  of  the  microscope  and  coin- 


FiG.  128.  Abbe  Camera  Lucida  in  Position.— -The 
prism  over  the  ocular  may  be  turned  aside  for  direct 
observation.  The  light  modifiers  for  drawing  surface 
and  microscope  are  in  connection  with  the  prism.  The 
prism  has  centering  screws  and  may  be  moved  up  or 
down  with  the  whole-  apparatus  by  the  clamping  ring 
around  the  top  of  the  draw-tube.  This  serves  to  place  the  prism  at  the  proper 
vertical  level  for  the  eye-point  of  different  oculars.  (Cut  loaned  by  the  Spen- 
cer Lens  Co.) 


cident  with  the  eye-point  of  the  ocular.  If  it  is  above  or  below,  or 
to  one  side  of  the  eye-point,  part  or  all  of  the,  field  of  the  microscope 
will  be  cut  off.  As  stated  above,  the  centering  screws  are  for  the 
proper  horizontal  arrangement  of  the  prism.  The  prism  is  set  at 
the  right  height  by  the  makers  for  the  eye-point  of  a  medium  ocular. 
If  one  desires  to  use  an  ocular  with  the  eye-point  farther  away  or 
nearer,  as  in  using  high  or  low  oculars  the  position  of  the  eye-point 
may  be  determined  as  directed  in  §  67  and  the  prism  loosened  and 


146  DRAWING  WITH  THE  MICROSCOPE  [  CH.   r 

raised  or  lowered  to  the  proper  level  ;  but  in  doing  this  one  should 
avoid  setting  the  prism  obliquely  to  the  mirror. 

In  the  latest  and  best  forms  of  this  camera  lucida  special 
arrangements  have  been  made  for  raising  or  lowering  the  prism  so 
that  it  may  be  used  with  equal  satisfaction  on  oculars  with  the  eye- 
point  at  different  levels,  and  the  prism  is  hinged  to  turn  aside  with- 
out disturbing  the  mirror  (Figs.  128,  132). 

One  can  determine  when  the  camera  is  in  a  proper  position  by 
looking  into  the  microscope  through  it.  If  the  field  of  the  micro- 
scope appears  as  a  circle  and  of  about  the  same  size  as  without  the 
camera  lucida,  then  the  prism  is  in  a  proper  position.  If  one  side 
of  the  field  is  dark,  then  the  prism  is  to  one  side  of  the  center  ;  if 
the  field  is  considerably  smaller  than  when  the  prism  is  turned  off 
the  ocular,  it  indicates  that  it  is  not  at  the  correct  level,  i.  <?.,  it  is 
above  or  below  the  eye-point. 

§  204.  Arrangement  of  the  Mirror  and  the  Drawing  Sur- 
face.— The  Abbe  camera  lucida  was  designed  for  use  with  a  vertical 
microscope  (Fig.  124).  On  a  vertical  microscope  if  the  mirror  is 
set  at  an  angle  of  45°,  the  axial  ray  is  at  right  angles  with  the  table 
top  or  a  drawing  board  which  is  horizontal,  and  a  drawing  made 
under  these  conditions  is  in  true  proportion  and  not  distorted.  The 
stage  of  most  microscopes,  however,  extends  out  so  far  at  the  sides 
that  with  a  45°  mirror  the  image  appears  in  part  on  the  stage  of  the 
microscope.  In  order  to  avoid  this  the  mirror  may  be  depressed  to 
some  point  below  45°,  say  at  40°  or  35°  (Fig.  129).  But  as  the 
axial  ray  from  the  mirror  to  the  prism  must  still  be  reflected  hori- 
zontally, it  follows  that  the.  axial  ray  no  longer  forms  an  angle  of 
90  degrees  with  the  drawing  surface,  but  a  greater  angle.  If  the 
mirror  is  depressed  to  35°,  then  the  axial  ray  takes  an  angle  of  110° 
with  a  horizontal  drawing  surface  (see  the  geometrical  figure  Fig. 
129  A).  To  make  the  angle  90°  again,  so  that  there  shall  be  no 
'distortion,  the  drawing  board  must  be  raised  "toward  the  microscope 
20°.  The  general  rule  is  to  raise  the  drawing  board  twice 
as  many  degrees  toward  the  microscope  as  the  mirror  is 
depressed  below  45°.  Practically  the  field  for  drawing  can 
always  be  made  free  of  the  stage  of  the  microscope,  at  45°,  at  40°, 
or  at  35°.  In  the  first  case  (45°  mirror)  the  drawing  surface  should 
be  horizontal,  in  the  second  case  (40°  mirror)  the  drawing  surface 
should  be  elevated  10°,  and  in  the  third  case  (35°  mirror)  the  draw- 


CH.    /']  DRAWING  WITH  THE  MICROSCOPE  147 

ing  board  should  be  elevated  20°  toward  the  microscope.  Further- 
more it  is  necessar)^  in  using  an  elevated  drawing  board  to  have  the 
mirror  bar  project  directly  laterally  so  that  the  edges  of  the  mirror 
are  in  planes  parallel  with  the  edges  of  the  drawing  board,  other- 
wise there  will  be  front  to  back  distortion,  although  the  elevation  of 
the  drawing  board  avoids  right  to  left  distortion.  If  one  has  a 
micrometer  ruled  in  squares  (net  micrometer}  the  distortion  pro- 
duced by  not  having  the  axial  ray  at  right  angles  with  the  drawing 
surface  may  be  very  strikingly  shown.  For  example,  set  the  mirror 
at  35°  and  use  a  horizontal  drawing  board.  With  a  pencil  make 
dots  at  the  corners  of  some  of  the  squares,  and  then  with  a  straight 
edge  connect  the  dots.  The  figures  will  be  considerably  longer 
from  right  to  left  than  from  front  to  back.  Circles  in  the  object 
appear  as  ellipses  in  the  drawings,  the  major  axis  being  from  right 
to  left. 

The  angle  of  the  mirror  may  be  determined  with  a  protractor, 
but  that  is  troublesome.  It  is  much  more  satisfactory  to  have  a 
quadrant  attached  to  the  mirror  and  an  indicator  on  the  projecting 
arm  of  the  mirror.  If  the  quadrant  is  graduated  throughout  its 
entire  extent,  or  preferably  at  three  points,  45°,  40°  and  35°,  one 
can  set  the  mirror  at  a  known  angle  in  a  moment,  then  the  drawing 
board  can  be  hinged  and  the  elevation  of  10°  and  20°  determined 
with  a  protractor.  The  drawing  board  is  very  conveniently  held  up 
by  a  broad  wedge.  By  marking  the  position  of  the  wedge  for  10° 
and  20°  the  protractor  need  be  used  but  once,  then  the  wedge  may 
be  put  into  position  at  any  time  for  the  proper  elevation. 

§  205.  Abbe  Camera  and  Inclined  Microscope.— It  is  very 
fatiguing  to  draw  continuously  with  a  vertical  microscope,  and  many 
mounted  objects  admit  of  an  inclination  of  the  microscope,  when 
one  can  sit  and  work  in  a  more  comfortable  position.  The  Abbe 
camera  is  as  perfectly  adapted  to  use  with  an  inclined  as  with  a 
vertical  microscope.  All  that  is  requisite  is  to  be  sure  that  the  fun- 
damental law  is  observed  regarding  the  axial  ray  of  the  image  and 
the  drawing  surface,  viz.,  that  they  should  be  at  right  angles.  This 
is  very  easily  accomplished  as  follows:  The  drawing  board  is  raised 
toward  the  microscope  twice  as  many  degrees  as  the  mirror  is  de- 
pressed below  45°  (§  204),  then  it  is  raised  exactly  as  many  degrees 
as  the  microscope  is  inclined,  and  in  the  same  direction,  that  is,  so 
the  end  of  the  drawing  board  shall  be  in  a  plane  parallel  with  the 


148 


DRAWING  WITH  THE  MICROSCOPE 


[  CH. 


FIG.  129 
Abbe  Camera  Lucida  in  position  to  avoid  distortion. 

FIG.  129.     The  Abbe  Camera  Lucida  with  the  mirror  at  35°. 

Axis,  Axis.   Axial  ray  from  the  microscope  and  from  the drawing  surj 'ace. 

A.  B.     Drawing  surface  raised  toward  the  microscope  20°. 

Foot.     The  foot  or  base  of  the  microscope. 

Mirror  with  quadrant  (Q).     The  mirror  is  seen  to  be  at  an  angle  0/55°. 

Ocular.     Ocular  of  the  Microscope. 

P.  P.     Drawing  pencil  and  the  cubical  prism  over  the  ocular. 

W*     Wedge  to  support  the  drawing  board. 

A.  Geometrical  figure  of  the  preceding ',  showing  the  angles  made  by  the 
axial  ray  ivith  the  mirror  and  the  necessary  elevation  of  the  drawing  board  to 
avoid  distortion.     From  the  equality  of  opposite  angles,  the  angle  of  the  a.vial 
ray  reflected  at  J5°  makes  an  angle  of  110°  zvith  a  horizontal  drawing  board. 
The  board  must  then  be  elevated  toivard  the  microscope-  20°  in  order  that  the 
axial  ray  may  be  perpendicular  to  it,  and  thus  fulfil  the  requirements  neces- 
sary to  avoid  distortion  (?  200,  204). 

B.  Upper  view  of  the  prism  of  the  camera  lucida.     A  considerable  por- 
tion of  the  face  of  the  prism  is  covered,  and  the  opening  in  the  silvered  surface 
appears  oval. 

C.  Quadrant  to  be  attached  to  the  mirror  of  the  Abbe  Camera  Lucida  to 
indicate  the  angle  of  the  mirror.     As  the  angle  is  nearly  always  45°,  40°  or 
J5°,  only  those  angles  are  shown. 


CIl.    l'\ 


DRAM' INC   ll'lTH  THE  MICROSCOPE 


149 


stage  of  the  microscope.     The  mirror  must  have  its  edges  in  planes 
parallel  with  the  edges  of  the  drawing  board  also  (Fig.  130.) 

§  206.  Drawing  with  the  Abbe  Camera  Lucida. — (A)  The 
light  from  the  microscope  and  from  the  drawing  surface  should  be 
of  nearly  equal  intensity,  so  that  the  image  and  the  drawing  pencil 
can  be  seen  with  about  equal  distinctness.  This  may  be  accomplished 


FIG.  130.  Bernhard's  Drawing  Board  for  the  Abbe  Camera  Lucida- 
This  draining  board  is  adjustable  vertically,  and  the  board  may  be  inclined  to 
prevent  distortion.  It  is  also  arranged  for  use  with  an  inclined  microscope, 
/taring  the  base  board  hinged,  Microscope  and  drawing  surface  are  then 
inclined  together.  (Zeit.  wiss.  Mikroskopie,  vol.  VII. ,  1894,  p.  208. )  (Zeiss 
Catalog. ) 

with  very  low  powers  (16  mm.  and  lower  objectives)  by  covering 
the  mirror  of  the  microscope  with  white  paper  when  transparent  ob- 
jects are  to  be  drawn.  For  high  powers  it  is  best  to  use  a  substage 
condenser.  Often  the  light  may  be  balanced  by  using  a  larger  or 
smaller  opening  in  the  diaphragm.  One  can  tell  which  field  is  ex- 
cessively illuminated,  for  it  is  the  one  in  which  objects  are  most  dis- 
tinctly seen.  If  it  is  the  microscopic,  then  the  image  of  the  micro- 


i5o  DRA  WING  WITH  THE  MICROSCOPE  [  CH.   V 

scopic  object  is  very  distinct  and  the  pencil  is  invisible  or  very  in- 
distinct. If  the  drawing  surface  is  too  brilliantly  lighted  the  pencil 
can  be  seen  clearly,  but  the  microscopic  image  is  obscure. 

When  opaque  objects,  that  is  objects  which  must  be  lighted 
with  reflected  light  (  §  72),  like  dark  colored  insects,  etc.,  are  to  be 
drawn  the  light  must  usually  be  concentrated  upon  the  object  in 
some  way.  The  microscope  may  be  placed  in  a  very  strong  light 
and  the  drawing  board  shaded  or  the  light  may  be  concentrated  upon 
the  object  by  means  of  a  concave  mirror  or  a  bull's  eye  condenser 
(Fig.  60). 

If  the  drawing  surface  is  too  brilliantly  illuminated,  it  may  be 
shaded  by  placing  a  book  or  a  ground  glass  screen  between  it  and 
the  window,  also  by  putting  one  or  more  smoked  glasses  in  the  path 
of  the  rays  from  the  mirror  (Fig.  1240).  If  the  light  in  the  mi- 
croscope is  too  intense,  it  may  be  lessened  by  using  white  paper 
over  the  mirror,  or  by  a  ground  glass  screen  between  the  microscope 
mirror  and  the  source  of  light  (Piersol,  Amer.  M.  M.  Jour.,  1888, 
p.  103).  It  is  also  an  excellent  plan  to  blacken  the  end  of  the  draw- 
ing pencil  with  carbon  ink.  Sometimes  it  is  easier  to  draw  on  a 
black  surface,  using  a  white  pencil  or  style.  The  carbon  paper 
used  in  manifolding  letters,  etc.,  may  be  used,  or  ordinary  black 
paper  may  be  lightly  rubbed  on  one  side  with  a  moderately  soft  lead 
pencil.  Place  the  black  paper  over  white  paper  and  trace  the  out- 
lines with  a  pointed  style  of  ivory  or  bone.  A  corresponding  dark 
line  will  appear  on  the  white  paper  beneath.  (  Jour.  Roy.  Micr.  Soc., 
1883,  p.  423). 

(A)  It  is  desirable  to  have  the  drawing  paper  fastened  with 
thumb  tacks,  or  in  some  other  way.  (B)  The  lines  made  while 
using  the  camera  lucida  should  be  very  light,  as  they  are  liable  to 
be  irregular.  (C)  Only  outlines  are  drawn  and  parts  located  with 
a  camera  lucida.  Details  are  put  in  free-hand.  (D)  It  is  some- 
times desirable  to  draw  the  outline  of  an  object  with  a  moderate 
power  and  add  the  details  with  a  higher  power.  If  this  is  done  it 
should  always  be  clearly  stated.  It  is  advisable  to  do  this  only 
with  objects  in  which  the  same  structure  is  many  times  duplicated, 
as  a  nerve  or  a  muscle.  In  such  an  object  all  the  different  struc- 
tures can  be  shown,  and  by  omitting  some  of  the  fibers  the  others 
may  be  made  plainer  without  an  undesirable  enlargement  of  the' 
entire  figure. 


CH.    V]  DRAWING  WITH  THE  MICROSCOPE  151 

(E)  If  a  drawing  of  a  given  size  is  desired  and  it  cannot  be 
obtained  by  any  combination  of  oculars,  objectives  and  lengths  of 
the  tube  of  the  microscope,  the  distance  between  the  camera  lucida 
and  the  table  may  be  increased  or  diminished  until  the  image  is  of 
the  desired  size.     This  distance  is  easily  changed  by   the  use  of  a 
book  or  a  block,  but  more  conveniently  if  one  has  a  drawing  board 
with  adjustable  drawing  surface  like  that  shown  in  Fig.  130. 

(F)  It  is  of  the  greatest  advantage,  as  suggested  by  Heinsius 
(Zeit.  w.  Mikr.,  1889,  p.  367),  to  have  the  camera  lucida  hinged  so 
that  the  prism  may  be  turned  off  the  ocular  for  a  moment's  glance 
at  the  preparation,  and  then  returned  in  place  without  the  necessity 
of  loosening  screws  and  readjusting  the  camera.     This  form   is  now 
made  by  several  opticians,  and  a  quadrant  is  added  by  some.      (Fig. 
128,  132.)     Any  skilled  mechanic  can  add  the  quadrant. 

§  207.  Magnification  of  the  Microscope  and  size  of 
Drawings  with  the  Abbe  Camera  Lucida. — In  determining  the 
standard  distance  of  250  millimeters  at  which  to  measure  the  image 
in  getting  the  magnification  of  the  microscope,  it  is  necessary  to 
measure  from  the  point  marked  P  on  the  prism  (Fig.  124)  to  the 
axis  of  the  mirror  and  then  vertically  to  the  drawing  board. 

In  getting  the  scale  to  which  a  drawing  is  enlarged  the  best 
way  is  to  remove  the  preparation  and  put  in  its  place  a  stage 
micrometer,  and  to  trace  a  few  (5  or  10)  of  its  lines  upon  one  corner 
of  the  drawing.  The  value  of  the  spaces  of  the  micrometer  being 
given,  thus  : 


,r>0th  mm. 

FIG.  131.     Showing  the  method  of  indicating  the  scale  at  which  a  drawing 
was  made. 

The  enlargement  of  the  figure  can  then  be  accurately  deter- 
mined at  any  time  by  measuring  with  a  steel  scale  the  length  of  the 
image  of  the  micrometer  spaces  and  dividing  it  by  their  known  size. 

Thus,  suppose  the  5  spaces  of  the  scale  of  enlargement  given 
with  a  drawing  were  found  to  measure  25  millimeters  and  the  spaces 
on  the  micrometer  were  T^  millimeter,  then  the  enlargement  is 
25-=-  yl^  =500.  That  is,  the  image  was  drawn  at  a  magnification 
of  500  diameters. 


152 


OR  AM' ING   \\'ITH  THE  MICROSCOPE 


\_Cff.    V 


If  the  micrometer  scale  is  added  to  every  drawing,  there  is  no 
need  of  troubling  one's  self  about  the  exact  distance  at  which  the 
drawing  is  made,  convenience  may  settle  that,  as  the  special  mag- 
nification in  each  case  may  be  determined  from  the  scale  accompany- 
ing the  picture.  It  should  be  remembered,  however,  that  the  con- 
ditions when  the  scale  is  drawn  must  be  exactly  as  when  the  draw- 
ing was  made. 


FIG.  132  A.  B.  Abbe  Camera  Lucida.  (A.}  In  this  figure  the  camera 
lucida  is  in  position  for  draining.  The  ring  or  collar  supporting  the  mirror 
is  graduated  so  that  the  angle  of  the  mirror-  may  be  exactly  determined. 
Smoked  glasses  serve  to  modify  the  light  from  the  microscope  or  from  the 
drawing  surface  as  needed.  By  means  of  a  clamping  ring  the  instrument  may 
b»  raised  or  lowered  to  accommodate  the  eye-point  in  dijferent  oculars. 

(B.)  In  this  figure  the  camera  lucida  prism  is  turned  back  so  that  one 
may  look  directly  into  the  ocular.  (Cuts  loaned  by  the  Bausch  &  Lomb  Opti- 
cal Co.) 

§  208.  Drawing  at  Slight  Magnification. — Some  objects  are 
of  considerable  size  and  for  drawings  should  be  enlarged  hut  a  few 
diameters, — 5  to  20.  By  using  sufficiently  low  objectives  and  differ- 
ent oculars  a  great  range  may  be  obtained.  Frequently,  however, 


CH. 


\\TTH  THE  MICROSCOPE 


153 


the  range  must  be  still  further  increased.  For  a  moderate  increase 
in  size  the  drawing  surface  may  be  put  farther  off  or,  as  one  more 
commonly  needs  less  rather  than  greater  magnification,  the  drawing 
surface  may  be  brought  nearer  the  mirror  of  the  camera  lucida  by 
piling  books  or  other  objects  on  the  drawing  board.  If  one  takes 
the  precaution  to  draw  a  scale  on  the  figure  under  the  same  condi- 
tions, its  enlargement  can  be  readily  determined  (§  207). 


FIG.  133.  Room  and  Apparatus  for  Drawing  with  the  Projection  Micro- 
scope. R.  Radiant,  an  arc  lamp  with  carbons  at  a  right  angle  ;  L.  t.  Lamp 
and  microscope  table ;  C.  Condenser  zv it h  W.  a  large  water  bath  between  the 
lenses  to  absorb  the  heat  rays;  S.  w.  Stage  and  stage  water  bath  on  which 
rests  the  object  and  keeps  the  object  cool  by  radiation  as  well  as  by  absorption  ; 
O.  The  objective  representing  the  microscope  ;  M.  Mirror  at  45°  on  a  draw- 
ing table,  (Dt.).  As  the  microscope  is  horizontal  so  that  the  axial  ray  is 
reflected  downward  at  right  angles  by  the  45°  mirror  there  is  no  distortion. 
The  scale  of  the  drawing  is  added  exactly  as  described  in  I  207. 

A  very  satisfactory  way  to  draw  at  low  magnifications  is  to  use 
a  simple  microscope  and  arrange  a  camera  lucida  over  it  as  over  the 
ocular.  In  this  way  one  may  get  drawings  at  almost  any  low  mag- 
nification. 


154  DRAWING   WITH  THE  MICROSCOPE  {CH.    V 

If  one  has  many  large  objects  to  draw  at  a  low  magnification, 
then  some  form  of  embryograph  is  very  convenient.  (Jour.  Roy. 
Micr.,  Soc.,  1899,  p.  223.)  The  writer  has  made  use  of  a  photo- 
graphic camera  and  different  photographic  objectives  for  the  purpose. 
The  object  is  illuminated  as  if  for  a  photograph  and  in  place  of  the 
ground  glass  a  plain  glass  is  used  and  on  this  some  tracing  paper  is 
stretched.  Nothing  is  then  easier  than  to  trace  the  outlines  of  the 
object.  See  also  Ch.  VIII. 

§209.  Drawing  with  the  Projection  Microscope. — Except 
for  the  highest  powers  and  for  details  of  cell  structure  the  projec- 
tion microscope  furnishes  the  most  satisfactory  means  of  making 
drawings.  With  it  one  can  draw  large  diagrams  or  small  figures 
directly  from  the  objects;  and  if  the  apparatus  is  properly  constructed 
one  may  make  diagrams  from  objects  60  to  70  mm.  in  diameter 
down  to  those  of  half  a  millimeter  or  less.  This  method  was  much 
in  vogue  and  highly  commended  by  the  older  microscopiste  who 
used  the  solar  microscope  (Baker,  Adams  and  Goring).  Since  the 
general  introduction  of  electric  lighting  drawing  with  the  projection 
microscope  has  become  once  more  common  and  is  the  most  satisfac- 
tory method  known  especially  for  the  numerous  drawings  necessary 
for  the  preparation  of  models  in  wax  or  blotting  paper.  See  Ch.  X. 

REFERENCES   FOR    CHAPTER   V 

Beale,  31,  355;  Behrens,  Kossel  and  Schiefferdecker,  77;  Carpeuter- 
Dallinger,  278  ;  VanHeurck,  91  ;  American  Naturalist,  1886,  p.  1071,  1887,  pp, 
1040-1043  ;  Amer.  Monthly  Micr.  Jour.,  1888,  p.  103  ;  1890,  p.  94  ;  Jour.  Roy. 
Micr.  Soc.,  1881,  p.  819,  1882,  p.  402,  1883,  pp.  283,  560,  1884,  p.  115,  1886,  p. 
516,  1888,  pp.  113,  809,  798;  Zeit.  wiss.  Mikroskopie,  1884,  pp.  1-21,  1889,  p. 
367,  1893,  pp.  289-295.  Here  is  described  an  excellent  apparatus  made  by 
Winkel.  Greenman  Anat.  Record  No.  7,  1907,  pp.  170-178.  Gage,  Origin  and 
Development  of  the  Projection  Microscope.  Transactions  of  the  Amer.  Micr. 
Soc.,  Vol.  XXVIII,  1906.  Consult  also  the  latest  catalogs  of  the  opticians. 


CHAPTER  VI 


MICRO-SPECTROSCOPE      AND     POLARISCOPE,     MICRO- 
CHEMISTRY,     MICRO-METALLOGRAPHY, 
TEXTILE     FIBERS 


APPARATUS   AND    MATERIAL    REQUIRED    FOR    THIS    CHAPTER 

Compound  microscope;  Micro-spectroscope  (§  210);  Watch-glasses  and 
shell  vials,  slides  and  covers  (§  229) ;  Various  substances  for  examination  (as 
blood  and  ammonium  sulphide,  permanganate  of  potash,  chlorophyll,  some 
colored  fruit,  etc.,  ($  230-239),  Micro-polarizer  (\  240);  Selenite  plate  ($  250); 
Various  doubly  refracting  objects,  as  crystals,  textile  fibers,  starch,  section  of 
bone;  Various  chemicals,  metals,  etc. 

MICRO-SPECTROSCOPE 

2  210.  A  Micro  Spectroscope,  Spectroscopic  or  Spectral  Ocular,  is  a  di- 
rect vision  spectroscope  in  connection  with  a  microscope  ocular.  The  one  de- 
vised by  Abbe  and  made  by  Zeiss  consists  of  a  direct  vision  spectroscope  prism 
of  the  Amici  pattern,  and  of  considerable  dispersion,  placed  over  the  ocular  of 
the  microscope.  This  direct  vision  or  Amici  prism  consists  of  a  single  trian- 
gular prism  of  heavy  flint  glass  in  the  middle  and  one  of  crown  glass'  on  each 
side,  the  edge  of  the  crown  glass  prisms  pointing  toward  the  base  of  the  flint 
glass  prism,  i.  e.,  the  edge  of  the  crown  and  flint  glass  prisms  point  in  oppo- 
site directions.  The  flint  glass  prism  serves  to  give  the  dispersion  or  separa- 
tion into  colors,  while  the  crown  glass  prisms  serve  to  make  the  emergent 
rays  approximately  parallel  with  the  incident  rays,  so  that  one  looks  directly 
into  the  prism  along  the  axis  of  the  microscope. 

The  Amici  prism  is  in  a  special  tube  which  is  hinged  to  the  ocular  and 
held  in  position  by  a  spring.  It  may  be  swung  free  of  the  ocular.  In  con- 
nection with  the  ocular  is  the  slit  mechanism  and  a  prism  for  reflecting  hori- 
zontal rays  vertically  for  the  purpose  of  obtaining  a  comparison  spectrum 
('4  223) .  Finally  near  the  top  is  a  lateral  tube  with  mirror  for  the  purpose  of 
projecting  an  Angstrom  scale  of  wave  lengths  upon  the  spectrum  ( \  224, 
Fig.  134-135). 

$  21 1.  Apparent  Reversal  of  the  Position  of  the  Colors  in  a  Direct  Vision 
Spectroscope. — In  accordance  with  the  statements  in  §  210  the  dispersion  or 
separation  into  colors  is  given  by  the  flint  glass  prism  or  prisms  and  in  ac- 


156  MICRO-SPECTROSCOPE  AND  POLARISCOPE    [  CH.    VI 


FIG.  134  Abbe's  Micro-spectroscope.  FIG.  135. 

Longitudinal  Section  of  Slit  Mechanism  separately, 

the  whole  instrument.  (Plan  view,  Full  size. ) 

(Y2  Full  size.) 

"  The  eye  lens  is  adjustable  so  as  to  accurately  focus  on  the  slit  situated  be- 
tween the  lenses.  The  mechanism  for  contracting  and  expanding  the  slit  is 
actuated  by  the  screw  F and  causes  the  laminae  to  move  symmetrically  (Merz's 
movement) .  The  slit  may  be  made  sufficiently  wide  so  as  to  include  the  whole 
.  visual  field.  The  screw  H  serves  to  limit  the  length  of  the  slit  so  as  to  com- 
pletely fill  the  latter  with  the  image  of  the  object  under  investigation  when  the 
comparison  prism  is  inserted.  The  comparison  prism  is  provided  with  a 
lateral  frame  and  clips  to  hold  the  object  and  the  illuminating  mirror.  All 
these  parts  are  encased  in  a  drum  on  the  ocular," 

"Above  the  eye-piece  is  placed  an  Amid  prism  of  great  dispersion  which 
may  be  turned  aside  about  the  pivot  A',  so  as  to  allow  of  the  adjustment  of  the 
object.  The  prism  is  retained  in  its  axial  position  by  the  spring  catch  L.  A 
scale  is  projected  on  the  spectrum  by  means  of  a  scale  tube  and  mirror  attached 
to  the  prism  casing.  The  divisions  of  the  scale  indicate  in  decimals  of  a  micron 
the  wave  length  of  the  respective  section  of  the  spectrum.  The  screw  P  serves 
to  adjust  the  scale  relative  to  the  spectrum." 

"  The  instrument  is  inserted  in  the  lube  in  place  of  the  ordinary  eye-piece 
and  is  clamped  to  the  former  by  means  of  the  screw  M  in  such  a  position  that 
the  mirrors  A  and  O,  ivhich  respectively  serve  to  illuminate  the  comparison 
prism  and  the  scale  of  wave  lengths  are  simultaneously  illuminated.1'1  (From 
Zeiss'  Catalog.) 


CH.   VI]     MICRO-SPECTROSCOPE  AND  POLARISCOPE 


157 


4     3  c 


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a 

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0 

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; 


I  II 


FIG.  136.  Various  Spectrums. — All  except  that  of  sodium  were  obtained 
by  diffused  day-light  with  the  slit  of  such  a  width  as  gave  the  most  distinct 
Fraunhofer  lines. 

It  frequently  occurs  that  with  a  substance  giving  several  absorption  bands 
(e.g.,  chlorophyll}  the  density  or  thickness  of  the  solution  must  be  varied  to 
show  all  the  different  bands  clearly. 

Solar  Spectrum. —  With  diffused  day-light  and  a  narrow  slit  the  spectrum 
is  not  visible  much  beyond  the  fixed  line  B.  In  order  to  extend  the  visible 
spectrum  in  the  red  to  the  line  A,  one  should  use  direct  sunlight  and  a  piece  of 
ruby  glass  in  place  of  the  ivatch-glass  in  Fig.  138. 

Sodium  Spectrum. — The  line  spectrum  ( \  213}  of  sodium  obtained  by  light- 
ing the  microscope  with  a  Bunsen  or  alcohol  flame  in  which  some  salt  of  sodium 
is  glowing.  With  the  micro-spectroscope  the  sodium  line  seen  in  the  solar 
spectrum  and  with  the  incandescent  sodium  appears  single,  except  under  very 
favorable  circumstances  ( \  214],  By  using  a  comparison  spectrum  of  day-light 
with  the  sodium  spectrum  the  light  and  dark  D-lines  will  be  seen  to  be  contin- 
uous as  here  shown. 

Permanganate  of  Potash. — This  spectrum  is  characterized  by  the  presence  of 
five  absorption  bands  in  the  middle  of  the  spectrum  and  is  best  shown  by  using 
a  r\j  per  cent,  solution  of  permanganate  in  water  in  a  -Match-glass  as  in  Fig.  sj8. 

Met-hemoglobin. — The  absorption  spectrum  of  met-hemoglobin  is  character- 
ized by  a  considerable  darkening  of  the  blue  end  of  the  spectrum  and  of  four 
Absorption  bands,  one  in  the  red  near  the  line  C  and  two  between  D  and  E 
nearly  in  the  place  of  the  two  bands  of  oxy-hemoglobin;  finally  there  is  a  some- 
what faint,  wide,  band  near  F.  Such  a  met-hemoglobin  spectrum  is  best  ob- 
tained by  making  a  solution  of  blood  in  water  of  such  a  concentration  that  the 
two  oxy-hcmoglobin  bands  run  together  (§  233},  and  then  adding  three  or  four 
drops  of  a  T'f  per  cent,  aqueous  solution  of  permanganate  of  potash  or  a  few 
drops  of  hydrogen  dioxid  (H^O^).  Soon  the  bright  red  will  change  to  a 
brownish  color,  when  it  may  be  examined. 


I58  MICRO-SPECTROSCOPE  AND  POLARISCOPE     [  CH.   VI 

cordance  with  the  general  law  that  the  waves  of  shortest  length,  blue,  etc., 
will  be  bent  most,  the  colors  have  the  position  indicated  in  the  top  of  Fig.  138, 
also  above  Fig.  134.  But  if  one  looks  into  the  direct  vision  spectroscope  or 
holds  the  eye  close  to  the  single  prism  (Fig.  139),  the  colors  will  appear  re- 
versed as  if  the  red  were  more  bent.  The  explanation  of  this  is  shown  in  Fig. 
139,  where  it  can  be  readily  seen  that  if  the  eye  is  placed  at  E,  close  to  the 
prism,  the  different  colored  rays  appear  in  the  direction  from  which  they 
reach  the  eye  and  consequently  are  crossed  in  being  projected  into  the  field 
of  vision  and  the  real  position  is  inverted.  The  same  is  true  in  looking  into 
the  micro-spectroscope.  The  actual  position  of  the  different  colors  may  be 
determined  by  placing  some  ground  glass  or  some  of  the  lens-paper  near  the 
prism  and  observing  with  the  eye  at  the  distance  of  distinct  vision.* 

VARIOUS    KINDS    OF   SPECTRA 

By  a  spectrum  is  meant  the  colored  bands  appearing  when  the  light 
traverses  a  dispersing  prism  or  a  diffraction  grating,  or  is  affected  in  any  way 
to  separate  the  diffierent  wave  lengths  of  light  into  groups.  When  daylight 
or  some  good  artificial  light  is  thus  dispersed  one  gets  the  appearance  so 
familiar  in  the  rainbow. 

\  212.  Continuous  Spectrum. — In  case  a  good  artificial  light  as  the  elec- 
tric light  is  used  the  various  rainbow  or  spectral  colors  merge  gradually  into 
one  another  in  passing  from  end  to  end  of  the  spectrum.  There  are  no  breaks 
or  gaps. 

g  213.  Line  Spectrum. — If  a  gas  is  made  incandescent,  the  spectrum  it 
produces  consists,  not  of  the  various  rainbow  colors,  but  of  sharp,  narrow, 
bright  lines,  the  color  depending  on  the  substance.  All  the  rest  of  the  spec- 
trum is  dark.  These  line  spectra  are  very  strikingly  shown  by  various  metals 
heated  to  incandescence. 

\  214.  Absorption  Spectrum. — By  this  is  meant  a  spectrum  in  which 
there  are  dark  lines  or  bands  in  the  spectrum.  The  most  striking  and  inter- 
esting of  the  absorption  spectra  is  the  Solar  Spectrum,  or  spectrum  of  sunlight. 
If  this  is  examined  by  a  good  spectroscope  it  will  be  found  to  be  crossed  by 
dark  lines,  the  appearance  being  as  if  one  were  to  draw  pen  marks  across  a 
continuous  spectrum  at  various  levels,  sometimes  apparently  between  the 
colors  and  sometimes  in  the  midst  of  a  color.  These  dark  lines  are  the  so- 
ca^led  Fraunhofer  Lines.  Some  of  the  principal  ones  have  been  lettered  with 
Roman  capitals,  A,  B;  C,  D,  E,  F,  G,  H,  commencing  at  the  red  end.  The 
meaning  of  these  lines  was  for  a  long  time  enigmatical,  but  it  is  now  known 
that  they  correspond  with  the  bright  lines  of  a  line  spectrum  (§213).  For 
example,  if  sodium  is  put  in  the  flame  of  a  spirit  or  Bunsen  lamp  it  will 
vaporize  and  become  luminous.  If  this  light  is  examined  there  will  be  seen 
one  or  two  bright  yellow  bands  corresponding  in  position  with  D  of  the  solar 

*The  author  wishes  to  acknowledge  the  aid  rendered  by  Professor  E.  L. 
Nichols  in  giving  the  explanation  offered  in  this  section. 


CH.   17]     MICRO-SPECTROSCOPE  AXD  POLARISCOPE 


J59 


spectrum  (Fig.  136).  If  now  the  spirit-lamp  flame,  colored  by  the  incan- 
descent sodium,  is  placed  in  the  path  of  the  electric  light,  and  it  is  examined 
as  before,  there  will  be  a  continuous  spectrum,  except  for  dark  lines  in  place 
of  the  bright  sodium  lines.  That  is,  the  comparatively  cool  yellow  light  of 
thespirit  lamp  cuts  off  or  absorbs  the  intensely  hot  yellow  light  of  the  electric 
light  ;  and  although  the  spirit  flame  sends  a  yellow  light  to  the  spectro- 
scope it  is  so  faint  in  comparison  with  the  electric  light  that  the  sodium  lines 
appear  dark.  It  is  believed  that  in  the  sun's  atmosphere  there  are  incan- 
descent metal  vapors  (sodium,  iron,  etc.),  but  that  they  are  so  cool  in  com- 
parison with  the  rays  of  their  wave  length  in  the  sun  that  the  cooler  light  of 
the  incandescent  metallic  vapors  absorb  the  light  of  corresponding  wave 
length,  and  are,  like  the  spirit  lamp-flame,  unable  to  make  up  the  loss,  and 
therefore  the  presence  of  the  dark  lines. 

2  215.  Absorption  Spectra  from  Colored  Substances. — While  the  solar 
spectrum  is  an  absorption  spectrum,  the  term  is  more  commonly  applied  to 
the  spectra  obtained  with  light  which  has  passed  through  or  has  been  reflected 
from  colored  objects  which  are  not  self-luminous. 

It  is  the  special  purpose  of  the  micro-spectroscope  to  investigate  the  spec- 
tra of  colored  objects  which  are  not  self-luminous,  i.  e. ,  blood  and  other 
liquids,  various  minerals,  as  monazite,  etc.  The  spectra  obtained  by  examin- 
ing the  light  reflected  from  these  colored  bodies  or  transmitted  through  them, 
possess,  like  the  solar  spectrum  dark  lines  or  bands,  but  the  bands  are  usually 
much  wider  and  less  sharply  defined.  Their  number  and  position  depend  on 
the  substance  or  its  constitution  (Fig.  137),  and  their  width,  in  part,  upon  the 
thickness  of  the  body.  With  some  colored  bodies,  no  definite  bands  are  pres- 
ent. The  spectrum  is  simply  restricted  at  one  or  both  ends  and  various  of  the 
other  colors  are  considerably  lessened  in  intensity.  This  is  true  of  many 
colored  fruits. 


FIG.  137.  Absorption  spectrum  of  Oxy-hemoglobin  or  arterial  blood  (/) 
and  of  Hemoglobin  or  venous  blood  (2).  (From  Gamgee  and  McMunn.) 

A,  B,  C,  D,  E,  F,  G,  H.  Some  of  the  Principal  Fraunhofer  lines  of  the 
solar  spectrum  (\  192). 

.00,  .80,  .70,  .60,  .50,  .40.  Wave  lengths  in  microns,  as  shown  in  Ang- 
strom's scale  (t  224}.  It  will  be  seen  that  the  wave  lengths  increase  toward  the 
red  and  decrease  toward  the  violet  end  of  the  spectrum . 

Red,  Yellow,  Orange,  etc.  Color  regions  of  the  spectrum.  Indigo  should 
come  between  the  blue  and  the  violet  to  complete  the  seven  colors  usually  given. 
It  was  omitted  through  inadvertence. 


160  .^HCRO-SPECTROSCOPE  AND  POLARISCOPE     [  CH.   VI 

£216.  Angstrom  and  Stokes'  Law  of  Absorption  Spectra — The  waves  of 
light  absorbed  by  a  body  when  light  is  transmitted  through  some  of  its  sub- 
stance are  precisely  the  waves  radiated  from  it  when  it  becomes  self-luminous. 
For  example,  a  piece  of  glass  that  is  yellow  when  cool,  gives  out  blue  light 
when  it  is  hot  enough  to  be  self-luminous.  Sodium  vapor  absorbs  two  bands 
of  yellow  light  (D  lines);  but  when  light  is  not  sent  through  it,  but  itself  is 
luminous  and  examined  as  a  source  of  light  its  spectrum  gives  bright  sodium 
lines,  all  the  rest  of  the  spectrum  being  dark  (Fig.  136). 

2  217.  Law  of  Color. — The  light  reaching  the  eye  from  a  colored,  solid, 
liquid  or  gaseous  body  lighted  with  white  light,  will  be  that  due  to  white  light 
less  the  light  waves  that  have  been  absorbed  by  the  colored  body.  Or  in  other 
words,  it  will  be  due  to  the  wave  lengths  of  light  that  finally  reach  the  eye 
from  the  object.  For  example,  a  thin  layer  of  blood  under  the  microscope 
will  appear  yellowish  green,  but  a  thick  layer  will  appear  pure  red.  If  now 
these  two  layers  are  examined  with  a  micro-spectroscope,  the  thin  layer  will 
show  all  the  colors,  but  the  red  end  will  be  slightly,  and  the  blue  end  consid- 
erably restricted,  and  some  of  the  colors  will  appear  considerably  lessened  in 
intensity.  Finally  there  may  appear  two  shadow-like  bands,  or  if  the  layer  is 
thick  enough,  two  well-defined  dark  bands  in  the  green  ($  232). 

If  the  thick  layer  is  examined  in  the  same  way,  the  spectrum  will  show 
only  red  with  a  little  orange  light,  all  the  rest  being  absorbed.  Thus  the 
spectroscope  shows  which  colors  remain,  in  part  or  wholly,  and  it  is  the  mix- 
ture of  this  remaining  or  unabsorbed  light  that  gives  color  to  the  object. 

\  218.  Complementary  Spectra. — While  it  is  believed  that  Angstrom's 
law  {\  216)  is  correct,  there  are  many  bodies  on  which  it  cannot  be  tested,  as 
they  change  in  chemical  or  molecular  constitution  before  reaching  a  suffi- 
ciently high  temperature  to  become  luminous.  There  are  compounds,  how- 
ever, like  those  of  didymium,  erbium  and  terbium,  which  do  not  change  with 
the  heat  necessary  to  render  them  luminous,  and  with  them  the  incandescence 
and  absorption  spectra  are  mutually  complementary,  the  one  presenting  bright 
lines  where  the  other  presents  dark  ones  (Daniell). 

ADJUSTING  THE   MICRO-SPECTROSCOPE 

§  219.  The  micro-spectroscope,  or  spectroscopic  ocular,  is  put 
in  the  place  of  the  ordinary  ocular  in  the  microscope,  and  clamped 
to  the  top  of  the  tube  by  means  of  a  screw  for  the  purpose. 

$  220.  Adjustment  of  the  Slit. — In  place  of  the  ordinary 
diaphragm  with  circular  opening,  the  spectral  ocular  has  a  dia- 
phragm composed  of  two  movable  knife  edges  by  which  a  slit-like 
opening  of  greater  or  less  width  and  length  may  be  obtained  at  will 
by  the  use  of  screws  for  the  purpose.  To  adjust  the  slit,  depress 
the  lever  holding  the  prism-tube  in  position  over  the  ocular,  and 


CH.   VI}     MICRO-SPECTROSCOPE  AND  POLARISCOPE  161 

swing  the  prism  aside.  One  can  then  look  into  the  ocular.  The 
lateral  screw  should  be  used  and  the  knife  edges  approached  till 
they  appear  about  half  a  millimeter  apart.  If  now  the  Amici  prism 
is  put  back  in  place  and  the  microscope  well  lighted,  one  will  see  a 
spectrum  by  looking  into  the  upper  end  of  the  spectroscope.  If  the 
slit  is  too  wide,  the  colors  will  overlap  in  the  middle  of  the  spectrum 
and  be  pure  only  at  the  red  and  blue  ends;  and  the  Fraunhofer  or 
other  bands  in  the  spectrum  will  be  faint  or  invisible.  Dust  on  the 
edges  of  the  slit  gives  the  appearance  of  longitudinal  streaks  on  the 
spectrum. 

§  221.  Mutual  Arrangement  of  Slit  and  Prism. — In  order 
that  the  spectrum  may  appear  as  if  made  up  of  colored  bands  going 
directly  across  the  long  axis  of  the  spectrum,  the  slit  must  be  paral- 
lel with  the  refracting  edge  of  the  prism.  If  the  slit  and  prism  are 
not  thus  mutually  arranged,  the  colored  bands  will  appear  oblique, 
and  the  whole  spectrum  may  be  greatly  narrowed.  If  the  colored 
bands  are  oblique,  grasp  the  prism  tube  and  slowly  rotate  it  to  the 
right  or  to  the  left  until  the  various  colored  bands  extend  directly 
across  the  spectrum. 

S  222.  Focusing  the  Slit.— In  order  that  the  lines  or  bands 
in  the  spectrum  shall  be  sharply  defined,  the  eye-lens  of  the  ocular 
should  be  accurately  focused  on  the  slit.  The  eye-lens  is  movable, 
and  when  the  prism  is  swung  aside  it  is  very  easy  to  focus  the  slit 
as  one  focused  for  the  ocular  micrometer  (§  172).  If  one  now  uses 
daylight  there  will  be  seen  in  the  spectrum  the  dark  Fraunhofer 
lines  (Fig.  136  E.  F.,  etc.). 

To  show  the  necessity  of  focusing  the  slit,  move  the  eye- lens 
down  or  up  as  far  as  possible,  and  the  Fraunhofer  lines  cannot  be 
seen.  While  looking  into  the  spectroscope  move  the  ocular  lens  up 
or  down,  and  when  it  is  focused  the  Fraunhofer  lines  will  reappear. 
As  the  different  colors  of  the  spectrum  have  different  wave  lengths, 
it  is  necessary  to  focus  the  slit  for  each  color  if  the  sharpest  possible 
pictures  are  desired. 

It  will  be  found  that  the  eye-lens  of  the  ocular  must  be  farther 
from  the  slit  for  the  sharpest  focus  of  the  red  end  than  for  the  sharp- 
est focus  of  the  lines  at  the  blue  end.  This  is  because  the  wave 
length  of  red  is  markedly  greater  than  for  blue  light. 

Longitudinal  dark  lines  of  the  spectrum  may  be  due  to  irregu- 


1 62 


MICRO-SPECTROSCOPE  AND  POLARISCOPE     [  CH.    VI 


i 


FIG.  138 


FIG.  139 


FIG.  140 


FIG.  138.  (i).  Section  of  the  tube  and  stage  of  the  microscope  with  the 
spectral  ocular  or  micro-spectroscope  in  position. 

Amid  Prism  ($  210). — The  direct  vision  prism  of  Amid  in  which  the  cen- 
tral shaded  prism  of  flint  glass  gives  the  dispersion  or  separation  into  colors, 
while  the  end  prisms  of  crown  glass  cause  the  rays  to  emerge  approximately 
parallel  with  the  axis  of  the  microscope.  A  single  ray  is  represented  as  enter- 
ing the  prism  and  this  is  divided  into  three  groups  (Red,  Yellow,  Blue] ,  which 


C/I.  VI]    MICRO-SPECTROSCOPE  AND  POLAR1SCOPE  163 

emerge  from  the  prism,  the  red  being  least  and  the  blue  most  bent  toward  the 
base  of  the  flint  prism  (see  Fig.  139). 

Hinge. —  The  hinge  on  which  the  prism  tube  turns  when  it  is  swung  off  the 
ocular. 

Ocular  (%  2 to) — The  ocular  in  which  the  slit  mechanism  takes  the  place  of 
the  diaphragm  ($220).  The.  eye-lens  is  movable  as  in  a  mici  ometcr  ocular,  so 
that  the  slit  may  be  accurately  focused  for  the  different  colors  (§  222). 

5".     Screw  for  setting  the  scale  of  wave  lengths  ($224). 

.V.     Screw/or  regulating  the  width  of  slit  ( £220). 

S'f.  Screw  for  clamping  the  micro-spectroscope  to  the  tube  of  the  micro- 
scope. 

Scale  Tube.  — The  tube  near  the  upper  end  containing  the  Angstrom  scale 
and  the  lenses  for  projecting  the  image  upon  the  upper  face  of  the  Amid  prism, 
whence  it  is  reflected  upward  to  the  eye  with  the  different  colored  rays.  At  the 
right  is  a  special  mirror  for  lighting  the  scale. 

Slit.  —  The  linear  opening  between  the  knife  edges.  Through  the  slit  the 
light  passes  to  the  prism.  It  must  be  arranged  parallel  with  the  refracting  edge 
of  the  prism,  and  of  such  a  width  that  the  Fraunhofer  or  Fixed  Lines  are  very 
clearly  and  sharply  defined  when  the  eye-lens  is  properly  fociised  (\  220-222) . 

Stage.  —  The  stage  of  the  microscope.  This  supports  a  watch-glass  with 
sloping  sides  for  containing  the  colored  liquid  to  be  examined. 

(3)  Comparison  Prism  with  tube  for  colored  liquid  (C.  L.),  and  mirror. 
The  prism  reflects  horizontal  rays  vertically,  so  that  when  the  prism  is  made  to 
cover  part  of the  slit  two  parallel  spectra  may  be  seen,   one  from  light  sent 
directly  through  the  entire  microscope  and  one  from  the  light  reflected  upward 
from  the  comparison  prism. 

(4)  'View  of  the  slit   mechanism  from   below. — Slit,   the   linear  space 
between  the  knife  edges  through  which  the  light  passes. 

P.     Comparison  prism  beneath  the  slit  and  covering  part  of  it  at  will. 

S.  S'.     Screws  for  regulating  the  length  and  width  of  the  slit. 

FIG.  139.  Flint-Glass  Prism  showing  the  separation  or  dispersion  of 
ichite  light  into  the  three  groups  of  colored  rays  (Red,  Yellow,  Blue),  the  blue 
rays  being  bent  the  most  from  the  refracting  edge  (£  211). 

FIG.  140.  Sectional  vierv  of  a  Microscope  with  the  Polariscope  in  position 
(I  240-242). 

Analyzer  and  Polarizer. — They  are  represented  with  corresponding  faces 
parallel  so  that  the  polarized  beam  could  traverse  freely  the  analyzer.  If 
either  Nicol  were  rotated  00°  they  would  be  crossed  and  no  light  would  traverse 
the  analyzer  unless  some  polarizing  substance  were  used  as  object,  (a)  Slot 
in  the  analyzer  tube  so  that  the  analyzer  may  be  raised  or  lowered  to  adjust  it 
for  difference  of  level  of  the  eye  point  in  different  oculars  (\  67,222). 

Pointer  and  Scale. — The  pointer  attached  to  the  analyzer  and  the  scale  or 
divided  circle  clamped  (by  the  screw  S)  to  the  tube  of  the  microscope.  The 
pointer  and  scale  enable  one  to  determine  the  exact  amount  of  rotation  of  the 
analyzer  (|  242). 

Object. —  The  object  whose  character  is  to  be  investigated  by  polarized  light. 


i64  MICRO-SPECTROSCOPE  AND  POLARISCOPE     [CH.    VI 

larity  of  the  edge  of  the  slit  or  to  the  presence  of  dust.      They  are 
most  troublesome  with  a  very  narrow  slit. 

§  223.  Comparison  or  Double  Spectrum. — In  order  to 
compare  the  spectra  of  two  different  substances  it  is  desirable  to  be 
able  to  examine  their  spectra  side  by  side.  This  is  provided  for  in 
the  better  forms  of  micro- spectroscopes  by  a  prism  just  below  the 
slit,  so  placed  that  the  light  entering  it  from  a  mirror  at  the  side  of 
the  drum  shall  be  totally  reflected  in  a  vertical  direction,  and  thus 
parallel  with  the  rays  from  the  microscope.  The  two  spectra  will 
be  side  by  side  with  a  narrow  dark  line  separating  them.  If  now 
the  slit  is  well  focused  and  daylight  be  sent  through  the  microscope 
and  into  the  side  to  the  reflecting  or  comparison  prism,  the  colored 
bands  and  the  Fraunhofer  dark  lines  will  appear  directly  continuous 
across  the  two  spectra.  The  prism  for  the  comparison  spectrum  is 
movable  and  may  be  thrown  entirely  out  of  the  field  if  desired. 
When  it  is  to  be  used,  it  is  moved  about  half  way  across  the  field  so 
that  the  two  spectra  shall  have  about  the  same  width. 

§  224.  Scale  of  Wave  Lengths. — In  the  Abbe  micro-spec-- 
troscope  the  scale  is  in  a  separate  tube  near  the  top  of  the  prism  and 
at  right  angles  to  the  prism-tube.  A  special  mirror  serves  to  light 
the  scale,  which  is  projected  upon  the  spectrum  by  a  lens  in  the 
scale- tube.  This  scale  is  of  the  Angstrom  form,  and  the  wave 
lengths  of  any  part  of  the  spectrum  may  be  read  off  directly,  after 
the  scale  is  once  set  in  the  proper  position,  that  is,  when  it  is  set  so 
that  any  given  wave  length  on  the  scale  is  opposite  the  part  of  the 
spectrum  known  by  previous  investigation  to  have  that  particular 
wave  length.  The  point  most  often  selected  for  setting  the  scale  is 
opposite  the  sodium  line  where  the  wave  length  is,  according  to 
Angstrom,  0.5892  //.  In  adjusting  the  scale,  one  may  focus  very 
sharply  the  dark  sodium  line  of  the  solar  spectrum  and  set  the  scale 
so  that  the  number  0.589  is  opposite  the  sodium  or  D  line,  or  a 
method  that  is  frequenty  used  and  serves  to  illustrate  §  213-214,  is 
to  sprinkle  some  salt  of  sodium  (carbonate  of  sodium  is  good)  in  a 
Bunsen  or  alcohol  lamp  flame  and  to  examine  this  flame.  If  this  is 
done  in  a  darkened  place  with  a  spectroscope,  a  narrow  bright  band 
will  be  seen  in  the  yellow  part  of  the  spectrum.  If  now  ordinary 
daylight  is  sent  through  the  comparison  prism,  the  bright  line  of 
the  sodium  will  be  seen  to  be  directly  continuous  with  the  dark  line 


CJ/.   VI]     MICRO-SPECTROSCOPE  AND  POLARISCOPE  165 

at  D  in  the  solar  spectrum  (Fig.  136).  By  reflecting  light  into  the 
scale-tube  the  image  of  the  scale  will  appear  on  the  spectrum,  and 
by  a  screw  just  under  the  scale-tube  but  within  the  prism-tube,  the 
proper  point  on  the  scale  (0.589/0  can  be  brought  opposite  the 
sodium  band.  All  the  scale  will  then  give  the  wave  lenghts  directly. 
Sometimes  the  scale  is  oblique  to  the  spectrum.  This  may  be 
remedied  by  turning  the  prism-tube  slightly  one  way  or  the  other. 
It  may  be  due  to  the  wrong  position  of  the  scale  itself.  If  so,  grasp 
the  milled  ring  at  the  distal  end  of  the  scale-tube  and,  while  looking 
into  the  spectroscope,  rotate  the  the  tube  until  the  lines  of  the  scale 
are  parallel  with  the  Fraunhofer  lines.  It  is  necessary  in  adjusting 
the  scale  to  be  sure  that  the  larger  number,  0.70,  is  at  the  red  end 
of  the  spectrum. 

The  numbers  on  the  scale  should  be  very  clearly  defined.  If 
they  do  not  so  appear,  the  scale-tube  must  be  focused  by  gasping 
the  outer  tube  of  the  scale-tube  and  moving  it  toward  or  from  the 
prism-tube  until  the  scale  is  distinct.  In  focusing  the  scale,  grasp 
the  outer  scale-tube  with  one  hand  and  the  prism-tube  with  the 
other,  and  push  or  pull  in  opposite  directions.  In  this  way  one  will 
be  less  liable  to  injure  the  spectroscope. 

§  225.  Designation  of  Wave  Length. — Wave  lengths  of 
light  are  designated  by  the  Greek  letter  A,  followed  by  the  number 
indicating  the  wave  length  in  some  fraction  of  a  meter.  With  the 
Abbe  microspectroscope  the  micron  is  taken  as  the  unit  as  with 
other  microscopical  measurements  (§  182).  Various  units  are  in 
use,  as  the  one  hundred  thousandth  of  a  millimeter,  millionths  or 
ten  millionths  of  a  millimeter.  If  these  smaller  units  are  taken,  the 
wave  lengths  will  be  indicated  either  as  a  decimal  fraction  of  a 
millimeter  or  as  whole  numbers.  Thus,  according  to  Angstrom, 
the  wave  length  of  sodium  light  is  5892  tenth  meters  or  Angstrom 
units,  or  5892  ten  millionths  mm.,  or  589.2  millionths,  or  58.92  one 
hundred  thousandths,  or  0.5892  one  thousandth  mm.,  or  0.5892^. 
The  last  would  be  indicated  thus,  A  0=0.5892  /<. 

§  226  Lighting  for  the  Micro-specftroscope. — For  opaque 
objects  a  strong  light  should  be  thrown  on  them  either  with  a  concave 
mirror  or  condensing  lens.  For  transparent  objects  the  amount  of 
the  substance  and  the  depth  of  color  must  be  considered.  As  a 
general  rule  it  is  well  to  use  plenty  of  light,  as  that  from  an  Abbe 


166  MICRO-SPECTROSCOPE  AND  POLAR1SCOPE     [  CH.    VI 

illuminator  with  a  large  opening  in  the  diaphragm  or  with  the 
diaphragm  entirely  open.  For  very  small  objects  and  thin  layers  of 
liquids  it  may  be  better  to  use  less  light.  One  must  try  both  meth- 
ods in  a  given  case,  and  learn  by  experience. 

The  direct  and  the  comparison  spectra  should  be  about  equally 
illuminated.  One  can  manage  this  by  putting  the  object  requiring 
the  greater  amount  of  illumination  on  the  stage  of  the  microscope 
and  lighting  it  with  the  Abbe  illuminator.  In  lighting  it  is  found 
in  general  that  for  red  or  yellow  objects,  lamp-light  gives  very  sat- 
isfactory results.  For  the  examination  of  blood  and  blood  crystals 
the  light  from  a  petroleum  lamp  is  excellent.  For  objects  with 
much  blue  or  violet,  daylight  or  artificial  light  rich  in  blue  light  is 
best. 

Furthermore,  one  should  be  on  his  guard  against  confusing  the 
ordinary  absorption  bands  with  the  Fraunhofer  lines  when  daylight 
is  used.  With  lamp-light  the  Fraunhofer  lines  are  absent  and, 
therefore,  not  a  source  of  possible  confusion. 

§  227.  Objective  to  Use  with  the  Micro-spectroscope.-— 
If  the  material  is  of  considerable  bulk,  a  low  objective  (16  to  50  mm.) 
is  to  be  preferred.  This  depends  on  the  nature  of  the  object  under 
examination,  however.  In  case  of  individual  crystals  one  should 
use  sufficient  magnification  to  make  the  real  image  of  the  crystal  en- 
tirely fill  the  width  of  the  slit.  The  length  of  the  slit  may  then  be 
regulated  by  the  screw  on  the  side  of  the  drum,  and  also  by  the 
comparison  prism.  If  the  object  does  not  fill  the  whole  slit  the 
white  light  entering  the  spectroscope  with  the  light  from  the  object 
might  obscure  the  absorption  bands.  For  opaque  objects  illumin- 
ating objectives  are  useful  (Fig.  143,  144).  • 

In  using  high  objectives  with  the  micro-spectroscope  one  must 
very  carefully  regulate  the  light  (Ch.  II)  and  sometimes  shade  the 
object. 

§  228.  Focusing  the  Objective. — For  focusing  the  objective 
the  prism- tube  is  swung  aside,  and  then  the  slit  made  wide  by  turn- 
ing the  adjusting  screw  at  the  side.  If  the  slit  is  open  one  can  see 
objects  when  the  microscope  is  focused  as  with  an  ordinary  ocular 
(§  220).  After  an  object  is  focused,  it  may  be  put  exactly  in  posi- 
tion to  fill  the  slit  of  the  spectroscope,  then  the  knife  edges  are 
brought  together  till  the  slit  is  of  the  right  width  ;  if  the  slit  is  then 


CH.   T/]    MICRO-SPECTROSCOPE  AND  POLARISCOPE  167 

too  long  it  may  be  shortened  by  using  one  of  the  mechanism  screws 
on  the  side,  or  if  that  is  not  sufficient,  by  bringing  the  comparison 
prism  farther  over  the  field.  If  one  now  replaces  the  Amici  prism 
and  looks  into  the  microscope,  the  spectrum  is  liable  to  have  longi- 
tudinal shimmering  lines.  To  get  rid  of  these  focus  up  or  down  a 
little  so  that  the  microscope  will  be  slightly  out  of  focus. 

§  229.  Amount  of  Material  Necessary  for  Absorption 
Spectra  and  its  Proper  Manipulation. — The  amount  of  material 
necessary  to  give  an  absorption  spectrum  varies  greatly  with  differ- 
ent substances,  and  can  be  determined  only  by  trial.  If  a  transpar- 
ent solid  is  under  investigation  it  is  well  to  have  it  in  the  form  of  a 
wedge,  then  successive  thicknesses  can  be  brought  under  the  micro- 
scope. If  a  liquid  substance  is  being  examined,  a  watch  glass  with 
sloping  sides  forms  an  excellent  vessel  to  contain  it,  then  successive 
thicknesses  of  the  liquid  can  be  brought  into  the  field  as  with  the 
wedge-shaped  solid.  Frequently  only  a  very  weak  solution  is  ob- 
tainable ;  in  this  case  it  can  be  placed  in  a  homoeopathic  vial,  or  in 
some  glass  tubing  sealed  at  the  end,  then  one  can  look  lengthwise 
through  the  liquid  and  get  the  effect  of  a  more  concentrated  solution. 
For  minute  bodies  like  crystals  or  blood  corpuscles,  one  may  proceed 
as  described  in  the  previous  section. 

MICRO-SPECTROSCOPE — EXPERIMENTS* 

§  230.  Put  the  micro-spectroscope  in  position,  arrange  the  slit 
and  the  Amici  prism  so  that  the  spectrum  will  show  the  various 
spectral  colors  going  directly  across  it  (§  220,  221)  and  focus  the 
slit.  This  may  be  done  either  by  swinging  the  prism  tube  aside 
and  proceeding  as  for  the  ocular  micrometer  (§  188),  or  by  moving 
the  eye-lens  of  the  ocular  up  and  down  while  looking  into  the  micro- 
spectroscope  until  the  dark  lines  of  the  solar  spectrum  are  distinct. 
If  they  cannot  be  made  distinct  by  focusing  the  slit,  then  the  light 
is  too  feeble  or  the  slit  is  too  wide  (§  220).  With  the  lever  move 
the  comparison  prism  across  half  the  field  so  that  the  two  spectra 
shall  be  of  about  equal  width.  For  lighting,  see  §  226. 

*If  one  does  not  possess  a  micro-spectroscope,  quite  satisfactory  results  may 
be  obtained  by  using  a  microscope  with  a  16  to  12  mm.  objective  and  a  pocket, 
direct  vision  spectroscope  in  place  of  the  eye-piece.  (Bleile,  Trans.  Amer. 
Micr.  Soc.  1900,  p.  8). 


i68  MICRO-SPECTROSCOPE  AND  POLAR1SCOPE    [  CH.   VI 

§  231.  Absorption  Spe<5ttrum  of  Permanganate  of  Pot- 
ash.— Make  a  solution  of  permanganate  of  potash  in  water  of  such 
a  strength  that  a  stratum  3  or  4  mm.  thick  is  transparent.  Put 
this  solution  in  a  watch-glass  with  sloping  sides,  and  put  it  under 
the  microscope.  Use  a  50  mm.  or  16  mm.  objective,  and  the 
full  opening  of  the  illuminator.  Light  strongly.  Look  into  the 
spectroscope  and  slowly  move  the  watch-glass  into  the  field.  Note 
carefully  the  appearance  with  the  thin  stratum  of  liquid  at  the  edge 
and  then  as  it  gradually  thickens  on  moving  the  watch-glass  still 
farther  along.  Count  the  absorption  bands  and  note  particularly 
the  red  and  blue  ends.  Compare  carefully  with  the  comparison 
spectrum  (Figs.  136,  137).  For  strength  of  solution  see  §  229. 

§  232.  Absorption  Spectrum  of  Blood. — Obtain  blood 
from  a  recently  killed  animal,  or  flame  a  needle,  and  after  it  is  cool 
prick  the  finger  two  or  three  times  in  a  small  area,  then  wind  a 
handkerchief  or  a  rubber  tube  around  the  base  of  the  finger,  and 
squeeze  the  finger  with  the  other  hand.  Some  blood  will  ooze  out 
of  the  pricks.  Rinse  this  off  into  a  watch-glass  partly  filled  with 
water.  Continue  to  add  the  blood  until  the  water  is  quite  red. 
Place  the  watch-glass  of  diluted  blood  under  the  microscope  in 
place  of  the  permanganate,  using  the  same  objective,  etc.  Note 
carefully  the  spectrum.  It  would  be  advantageous  to  determine  the 
wave  length  opposite  the  center  of  the  dark  bands.  This  may 
easily  be  done  by  setting  the  scale  properly  as  described  in  §  224. 
Make  another  preparation,  but  use  a  homeopathic  vial  instead  of  a 
watch-glass.  Cork  the  vial  and  lay  it  down  upon  the  stage  of  the 
microscope.  Observe  the  spectrum.  It  will  be  like  that  in  the 
watch-glass.  Remove  the  cork  and  look  through  the  whole  length 
of  the  vial.  The  bands  will  be  much  darker,  and  if  the  solution  is 
thick  enough  only  red  and  a  little  orange  will  appear.  Re- insert 
the  cork  and  incline  the  vial  so  that  the  light  traverses  a  very  thin 
layer,  then  gradually  elevate  the.  vial  and  the  effect  of  a  thicker  and 
thicker  layer  may  be  seen.  Note  especially  that  the  two  character- 
istic bauds  unite  and  form  one  wide  band  as  the  stratum  of  liquid 
thickens.  Compare  with  the  following  : 

Add  to  the  vial  of  diluted  blood  a  drop  or  two  of  ammonium 
sulphide,  such  as  is  used  for  a  reducing  agent  in  chemical  labora- 
tories. Shake  the  bottle  gently  and  then  allow  it  to  stand  for  ten 
or  fifteen  minutes.  Examine  it  and  the  two  bands  will  have  been 


CH.    VII     MICRO-SPECTROSCOPE  AXD  POLARISCOPE  169 

replaced  by  a  single,  less  clearly  defined  band  in  about  the  same 
position.  The  blood  will  also  appear  somewhat  purple.  Remove 
the  cork  to  admit  fresh  air  then  shake  the  vial  vigorously  and  the 
color  will  change  to  the  bright  red  of  fresh  blood.  Examine  it 
again  with  the  spectroscope  and  the  two  bands  will  be  visible. 
After  five  or  ten  minutes  another  examination  will  show  but  a 
single  band.  Incline  the  bottle  so  that  a  thin  stratum  may  be 
examined.  Note  that  the  stratum  of  liquid  must  be  considerably 
thicker  to  show  the  absorption  band  than  was  necessary  to  show 
the  two  bands  in  the  first  fexperjment.  Furthermore,  while  the 
single  band  ma)'  be  made  quite  black  on  thickening  the  stratum,  it 
will  not  separate  into  two  bands  with  a  thinner  stratum.  In  this 
experiment  it  is  very  instructive  to  have  the  watch-glass  of  arterial 
blood  under  the  microscope  and  the  vial  of  blood  to  which  has  been 
added  the  ammonium  sulphide  in  position  for  a  comparison 
spectrum. 

The  two  banded  spectrum  is  that  of  oxy- hemoglobin,  or  arterial 
blood,  the  single  banded  spectrum  of  hemoglobin  (sometimes  called 
reduced  hemoglobin)  or  venous  blood,  that  is,  the  respiratory  oxy- 
gen is  present  in  the  two  banded  spectrum  but  absent  from  the 
single  banded  spectrum.  When  the  bottle  was  shaken  the  hemo- 
globin took  up  oxygen  from  the  air  and  became  oxy-hemoglobin,  as 
occurs  in  the  lungs,  but  soon  the  ammonium  sulphide  took  away 
the  respiratory  oxygen,  thus  reducing  the  oxy-hemoglobin  to 
hemoglobin.  This  may  be  repeated  many  times  (Fig.  137). 

£  233.  Met-Hemoglobin. — The  absorption  spectrum  of  met- 
hemoglobin  is  characterized  by  a  considerable  darkening  of  the  blue 
end  of  the  spectrum  and  of  four  absorption  bands,  one  in  the  red 
near  the  line  C  and  two  between  D  and  E,  nearly  in  the  place  of 
the  two  bands  of  oxy-hemoglobin  ;  finally  there  is  a  somewhat  faint, 
wide  band  near  F.  Such  a  met-hemoglobin  spectrum  is  best 
obtained  by  making  a  solution  of  blood  in  water  of  such  a  concen- 
tration that  the  two  oxy-hemoglobin  bands  run  together,  and  then 
adding  three  or  four  drops  of  a  ^  per  cent  aqueous  solution  of  per- 
manganate of  potash.  Soon  the  bright  red  will  change  to  a  brown- 
ish color,  when  it  may  be  examined  (Fig.  136).  Instead  of  the 
permanganate  one  may  use  hydrogen  dioxide  (H2O2). 

>j  234.      Carbon  Monoxide  Hemoglobin  (CO-Hemoglobin).— 


170  MICRO-SPECTROSCOPE  AND  POLARISCOPE     [  CH.    VI 

To  obtain  this,  kill  an  animal  in  illuminating  gas,  or  one  may  allow 
illuminating  gas  to  bubble  through  some  blood  already  taken  from 
the  body.  The  gas  should  bubble  through  a  minute  or  two.  The 
oxygen  will  be  displaced  by  carbon  monoxide.  This  forms  quite  a 
stable  compound  with  hemoglobin,  and  is  of  a  bright  cherry- red 
color.  Its  spectrum  is  nearly  like  that  of  oxy-hemoglobin,  but  the 
bands  are  farther  toward  the  blue.  Add  several  drops  of  ammonium 
sulphide  and  allow  the  blood  to  stand  some  time.  No  reduction 
will  take  place,  thus  forming  a  marked  contrast  to  solutions  of  oxy- 
hemoglobin.  By  the  addition  of  a  few' drops  of  glacial  acetic  acid  a 
dark  brownish  red  color  is  produced. 

§  235-  Carmine  Solution. — Make  a  solution  of  carmine  by 
putting  y'jy  gram  of  carmine  in  100  cc.  of  water  and  adding  10  drops 
of  strong  ammonia.  Put  some  of  this  in  a  watch-glass  or  in  a  small 
vial  and  compare  the  spectrum  with  that  of  oxy-hemoglobin  or  car- 
bon monoxide  hemoglobin.  It  has  two  bands  in  nearly  the  same 
position,  thus  giving  the  spectrum  a  striking  similarity  to  blood. 
If  now  several  drops,  15  or  20,  of  glacial  acetic  acid  are  added  to 
the  carmine,  the  bands  remain  and  the  color  is  not  markedly 
changed,  while  with  either  oxy-hernoglobin  or  CO-hemoglobin  the 
color  is  decidedly  changed  from  the  bright  red  to  a  dull  reddish 
brown,  and  the  spectrum,  if  any  can  be  seen,  is  markedly  different. 
Carmine  and  O-hemoglobin  can  be  distinguished  by  the  use  of 
ammonium  sulphide,  the  carmine  remaining  practically  unchanged 
while  the  blood  shows  the  single  band  of  hemoglobin  (§  232).  The 
acetic  acid  serves  to  differentiate  the  CO-hemoglobin  as  well  as  the 
O-hemoglobin. 

§  236.  Colored  Bodies  not  giving  Distinctly  Banded 
Absorption  Specftra. — Some  quite  brilliantly  colored  objects,  like 
the  skin  of  a  red  apple,  do  not  give  a  banded  spectrum.  Take  the 
skin  of  a  red  apple,  mount  it  on  a  slide,  put  on  a  cover-glass  and 
add  a  drop  of  water  at  the  edge  of  the  cover.  Put  the  preparation 
under  the  microscope  and  observe  the  spectrum.  Although  no  bands 
will  appear,  in  some  cases  at  least,  yet  the  ends  of  the  spectrum  will 
be  restricted  and  various  regions  of  the  spectrum  will  not  be  so 
bright  as  the  comparison  spectrum.  Here  the  red  color  arises  from 
the  mixture  of  the  unabsorbed  waves,  as  occurs  with  other  colored 
objects.  In  this  case,  however,  not  all  the  light  of  a  given  wave 
length  is  absorbed,  consequently  there  are  no  clearly  defined  dark 


CH.    /'/]     MICRO-SPECTROSCOPE  AND  POLARISCOT1  171 

bands,  the  light  is  simply  less  brilliant   in   certain   regions  and   the 
red  rays  so  predominate  that  they  give  the  prevailing  color. 

§  237.  Nearly  Colorless  Bodies  with  Clearly  Marked 
Absorption  Spectra. — In  contradistinction  to  the  brightly  colored 
objects  with  no  distinct  absorption  bauds  are  those  nearly  colorless 
bodies  and  solutions  which  give  as  sharply  defined  absorption  bands 
as  could  be  desired.  The  best  examples  of  this  are  afforded  by 
solutions  of  the  rare  earths,  didyinium,  etc.  These  in  solutions 
that  give  hardly  a  trace  of  color  to  the  eye  give  absorption  bands 
that  almost  rival  the  Fraunhofer  lines  in  sharpness. 

S  238.  Absorption  Spectra  of  Minerals. — As  example  take 
some  monazite  sand  on  a  slide  and  either  mount  it  in  balsam  (see 
Ch.  IX),  or  cover  and  add  a  drop  of  water.  The  examination  may 
be  made  also  with  the  dry  sand,  but  it  is  less  satisfactory.  Light 
well  with  transmitted  light,  and  move  the  preparation  slowly 
around.  Absorption  bands  will  appear  occasionally.  Swing  the 
prism  tube  off  the  ocular,  open  the  slit  and  focus  the  sand.  Get  the 
image  of  one  or  more  grains  directly  in  the  slit,  then  narrow  and 
shorten  the  slit  so  that  no  light  can  reach  the  spectroscope  that  has 
not  traversed  the  grain  of  sand.  The  spectrum  will  be  satisfactory 
under  such  conditions.  It  is  frequently  of  great  service  in  deter- 
mining the  character  of  unknown  mineral  sands  to  compare  the 
spectra  with  known  minerals.  If  the  absorption  bands  are  identical, 
it  is  strong  evidence  in  favor  of  the  identity  of  the  minerals.  For 
proper  lighting  see  §  226. 

§  239.  While  the  study  ot  absorption  spectra  gives  one  a 
great  deal  of  accurate  information,  great  caution  must  be  exercised 
in  drawing  conclusions  as  to  the  identity  or  even  the  close  relation- 
ship of  bodies  giving  approximately  the  same  absorption  spectra. 
The  rule  followed  by  the  best  workers  is  to  have  a  known  body  as 
control  and  to  treat  the  unknown  body  and  known  body  with  the 
same  reagents,  and  to  dissolve  them  in  the  same  medium.  If  all 
the  reactions  are  identical  then  the  presumption  is  strong  that  the 
bodies  are  identical  or  very  closely  related.  For  example,  while 
one  might  be  in  doubt  between  a  solution  of  oxy-  or  CO-hemoglobin 
and  carmine,  the  addition  of  ammonium  sulphide  serves  to  change 
the  double  to  a  single  band  in  the  O-hemoglobin,  and  glacial  acetic 
acid  enables  one  to  distinguish  between  the  CO-blood  and  the  car- 


172  MICRO-SPECTROSCOPE  AND  POLARISCOPE    [  CH.   VI 

mine,  although  the  ammonium  sulphide  would  not  enable  one  to 
make  the  distinction.  Furthermore  it  is  unsafe  to  compare  objects 
dissolved  in  different  media.  Different  objects  as  "  cyanine  and 
aniline  blue  dissolved  in  alcohol  give  a  very  similar  spectrum,  but 
in  water  a  totally  different  one."  "Totally  different  bodies  show 
absorption  bands  in  exactly  the  same  position  (solid  nitrate  of  ura- 
nium and  permanganate  of  potash  in  the  blue)."  (MacMunn). 
The  rule  given  by  MacMunn  is  a  good  one  :  "  The  recognition  of 
a  body  becomes  more  certain  if  its  spectrum  consists  of  several 
absorption  bands,  but  even  the  coincidence  of  these  bands  with 
those  of  another  body  is  not  sufficient  to  enable  us  to  infer  chemical 
identity  ;  what  enables  us  to  do  so  with  certainty  is  the  fact  :  that 
the  two  solutions  give  bands  of  equal  intensities  in  the  same  parts  oj  the 
spectrum  which  undergo  analogous  changes  on  the  addition  of  the  same 
reagent. ' ' 

REFERENCES    TO    THE    MICRO-SPECTROSCOPE    AND 
SPECTRUM    ANALYSIS 

The  micro-spectroscope  is  playing  an  ever-increasingly  important  role  in 
the  spectrum  analysis  of  animal  and  vegetable  pigments,  and  of  colored 
mineral  and  chemical  substances,  theiefore  a  somewhat  extended  reference  to 
literature  is  given.  Full  titles  of  the  books  and  periodicals  will  be  found  in 
the  Bibliography  at  the  end. 

Angstrom,  Recherches  sur  le  spectre  solaire,  etc.  Also  various  papers  in 
periodicals.  See  Royal  Soc's  Cat'l  Scientific  Papers;  Anthony  &  Brackett  ; 
Beale,  p.  269  ;  Behrens,  p.  139  ;  Kossel  und  Schiefferdecker,  p.  63  ;  Carpenter, 
p.  323  ;  Browning,  How  to  Work  with  the  Spectroscope,  and  in  Monthly  Micr. 
Jour.,  II,  p.  65  ;  Daniell,  Principles  of  Physics.  The  general  principles  of 
spectrum  analysis  are  especially  well  stated  in  this  work,  pp.  435-455  ;  Davis, 
p.  342;  Dippel,  p.  277  ;  Frey  ;  Gamgee,  p.  91  ;  Halliburton  ;  Hogg,  p,  122  ; 
also  in  Monthly  Micr.  Jour.,  Vol.  II,  on  colors  of  flowers;  Jour.  Roy.  Micr. 
Soc. ,  1880,  1883,  and  in  various  other  vols. ;  Kraus;  Lockyer;  M'Kendrick; 
MacMunn;  and  also  in  Philos,  Trans.  R.  S.,  1886;  various  vols.  of  Jour  Physiol.; 
Nageli  uiid  Schwendener;  Proctor;  Ref.  Hand-Book  Med.  Science,  Vol.  I,  p. 
577,  VI.  p.  516,  VII,  p.  426;  Roscoe;  Schellen;  Sorby,  in  Beale,  p.  269;  also 
Proc.  R.  S.,  1874,  p.  31,  1867,  p.  433;  see  also  in  the  Scientific  Review,  Vol. 
V,  p.  66,  Vol.  II,  p.  419  ;  Landauer,  Spectrum  Analysis.  The  larger  works  on 
Physiology,  Chemistry  and  Physics  may  also  be  consulted  with  profit. 

Vogel,  Spectrum  analysis;  also  in  Nature,  Vol.  xix,  p.  495,  on  absorption 
spectra.  The  bibliography  in  MacMunn  is  excellent  and  extended. 

For  hemochromogen  in  medico-legal  cases  see  Bleile,  Trans.  Aruer.  Micr. 
Soc.,  1900,  p.  9. 


CH.   VI}     MICRO-SPECTROSCOPE  AND  POLARISCOPE  173 

MICRO-POLARISCOPE 

\  240.  The  tnicro-polariscope,  or  polarizer,  is  a  polariscope  used  in  con- 
nection with  a  microscope. 

The  most  common  and  typical  form  consists  of  two  Nicol  prisms,  that  is, 
two  somewhat  elongated  rhombs  of  Iceland  spar  cut  diagonally  and  cemented 
together  with  Canada  balsam.  These  Nicol  prisms  are  then  mounted  in  such 
a  way  that  the  light  passes  through  them  lengthwise,  and  in  passing  is  divided 
into  two  rays  of  plane  polarized  light.  The  one  of  these  rays  obeying  the 
ordinary  law  of  refraction  is  called  the  ordinary  ray,  the  one  departing  from 
the  law  is  called  the  e.vtra-ordinary  ray.  These  two  rays  are  polarized  in 
planes  at  right  angles  to  each  other.  The  Nicol  prism  totally  reflects  the 
ordinary  ray  at  the  cemented  surface  as  it  meets  that  surface  at  an  angle 
greater  than  the  critical  angle,  and  only  the  less  refracted  extraordinary  ray 
is  transmitted. 

</  241.  Polarizer  and  Analyzer. — The  polarizer  is  one  of  the  Nicol  prisms. 
It  is  placed  beneath  the  object  and  in  this  way  the  object  is  illuminated  with 
polarized  light.  The  analyzer  is  the  other  Nicol  and  is  placed  at  some  level 
above  the  object, very  conveniently  above  the  ocular. 

When  the  corresponding  faces  of  the  polarizer  and  analyzer  are  parallel 
/.  e.,  when  the  faces  through  which  the  oblique  section  passes  are  parallel, 
light  passes  freely  through  the  analyzer  to  the  eye.  If  these  corresponding 
faces  are  at  right  angles,  that  is,  if  the  Nicols  are  crossed,  then  the  light  is  en- 
tirely cut  off  and  the  two  transparent  prisms  become  opaque  to  ordinary  light. 
There  are  then,  in  the  complete  revolution  of  the  analyzer,  two  points  at  o° 
and  180°,  where  the  corresponding  faces  are  parallel  and  where  light  freely 
traverses  the  analyzer.  There  are  also  two  crossing  points  of  the  Nicols,  at 
90°  and  270°,  where  the  light  is  extinguished.  In  the  intermediate  points 
there  is  a  sort  of  twilight. 

\  242.  Putting  the  Polarizer  and  Analyzer  in  Position. — Swing  the  dia- 
phragm carrier  of  the  Abbe  illuminator  out  from  under  the  illuminator, 
remove  the  disk  diaphragm  or  open  widely  the  iris  diaphragm  and  place  the 
analyzer  in  the  diaphragm  carrier,  then  swing  it  back  under  the  illuminator. 
Remove  the  ocular,  put  the  graduated  ring  on  the  top  of  the  tube  and  then 
replace  the  ocular  and  put  the  analyzer  over  the  ocular  and  ring.  Arrange  the 
graduated  ring  so  that  the  indicator  shall  stand  at  o°  when  the  field  is  lightest. 
This  may  be  done  by  turning  the  tube  down  so  that  the  objective  is  near  the 
illuminator,  then  shading  the  stage  so  that  none  but  polarized  light  shall  enter 
the  microscope.  Rotate  the  analyzer  until  the  lightest  possible  point  is  found, 
then  rotate  the  graduated  ring  till  the  index  stands  at  o°.  The  ring  may  then 
be  clamped  to  the  tube  by  the  side  screw  for  the  purpose.  Or,  more  easily, 
one  may  set  the  index  at  o°,  clamp  the  ring  to  the  microscope,  then  rotate  the 
draw-tube  of  the  microscope  till  the  field  is  lightest. 

£243.  Adjustment  of  the  Analyzer. — The  analyzer  should  be  capable  of 
moving  up  and  down  on  its  mounting,  so  that  it  can  be  adjusted  to  the  eye- 


174  MICRO-SPECTROSCOPE  AND  POLARISCOPE     [  Cff.   V[ 

point  of  the  ocular  with  which  it  is  used.  If  on  looking  into  theanalyzer  with 
parallel  Nicols  the  edge  of  the  field  is  not  sharp,  or  if  it  is  colored,  the  analyz- 
er is  not  in  proper  position  with  reference  to  the  eye  point,  and  should  be 
raised  or  lowered  till  the  edge  of  the  field  is  perfectly  sharp  and  as  free  from 
color  as  the  ocular  itself  is  when  the  analyzer  is  removed. 

\  244.  Objectives  to  Use  with  the  Polariscope. — Objectives  of  all  powers 
may  be  used,  including  the  homogenous  immersion.  In  general,  however,  the 
lower  powers  are  somewhat  more  satisfactory.  A  good  rule  to  follow  in  this 
case  is  the  general  rule  in  all  microscopic  work, — use  the  power  that  most  clear- 
ly and  satisfactorily  sJiows  the  object  under  investigation. 

$  245.  Lighting  for  Micro-Polariscope  Work. — Follow  the  general  direc- 
tions given  in  Chapter  II.  It  is  especially  necessary  to  shade  the  object  so 
that  no  unpolarized  light  can  enter  the  objective,  otherwise  the  field  cannot  be 
sufficiently  darkened.  No  diaphragm  is  used  over  the  polarizer  for  most  exam- 
inations. Direct  sunlight  may  be  used  to  advantage  with  some  objects,  and 
the  object  should  be  as  transperent  as  possible. 

\  246.  Mounting  Objects  for  the  Polariscope. — So  far  as  possible  objects 
should  be  mounted  in  balsam  to  render  them  transparent.  In  many  cases 
objects  mounted  in  water  do  not  give  satisfactory  appearances  with  the  polar- 
iscope.  For  example,  if  starch  is  mounted  dry  in  water,  the  appearances  are 
not  so  striking  as  if  mounted  in  balsam  (Davis,  p.  337  ;  Suffolk). 

§  247.  Purpose  of  a  Micro-Polasiscope. —  (A)  To  determine  whether  a 
microscopic  object  is  singlv  or  doubly  refractive,  i.  e.  isotropic  or  anisotropic. 
(B)  To  determine  whether  or  not  a  body  shows  pleochroism.  (C)  To  show 
whether  an  object  rotates  the  plane  of  polarization,  as  with  sugar.  (D)  To 
give  beautiful  colors. 

For  petrological  and  mineralogical  investigations  the  microscope  should 
possess  a  graduated,  rotating  stage  so  that  the  object  can  be  rotated,  and  the 
exact  angle  of  rotation  determined.  It  is  also  found  of  advantage  in  investi- 
gating objects  with  polarized  light  where  colors  appear,  to  combine  a  polar- 
iscope  and  spectroscope  (Spectro-Polariscope). 

MICRO-POLARISCOPE — EXPERIMENTS 

§  248-.  Arrange  the  polarizer  and  anlyzer  as  directed  above 
(§  242)  and  use  a  16  mm.  objective  except  when  otherwise  directed. 

(A)"  Isotropic  or  Singly  Refracting  Objects. — Light  the 
microscope  well  and  cross  the  Nicols,  shade  the  stage  and  make  the 
field  as  dark  as  possible  (§  241).  For  an  isotropic  substance,  put 
an  ordinary  glass  slide  under  the  microscope.  The  field  will  remain 
dark.  As  an  example  of  crystals  belonging  to  the  cubical  system 
and  hence  isotropic,  make  a  strong  solution  of  common  salt  (sodium 
chlorid)  put  a  drop  on  a  slide  and  allow  it  to  crystallize, 


CH.    /Y]     MICRO-SPECTROSCOPE  AND  POLARrSCOPE  I75 

put  it  under  the  microscope,  remove  the  analy/.er,  focus  the  crystals 
and  then  replace  the  analyzer  and  cross  the  Nicols.  The  field  and 
the  crystals  will  remain  dark. 

(B)  Anisotropic  or  Doubly  Refracting  Objects. — Make  a 
fresh  preparation  of  carbonate  of  lime  crystals   like    that    described 
for  pedesis  (§  164),  or  use  a  preparation  in  which  the  crystals  have 
dried  to  the  slide,  use  a  5  or  3   mm.    objective,   shade   the   object 
well,  remove  the  analyzer  and  focus  the  crystals,  then  replace  the 
analyzer.     Cross  the  Nicols.     In  the  dark  field  will  be  seen  multi- 
tudes of  shining  crystals,   and  if  the  preparation  is  a  fresh  one  in 
water,  part  of  the  smaller  crystals  will  alternately  flash  and  disap- 
pear.    By  observing  carefully,  some  of  the  larger  crystals  will  be 
found  to  remain  dark  with  crossed  Nicols,  others  will  shine  contin- 
uously.    If  the  crystals  are  in  such  a  position  that  the  light  passes 
through  them  parallel  with  the  optic  axis,*  the  crystals  are  isotropic 
like  salt  crystals  and  remain  dark.     If,  however,  the  light  traverses 
them  in  any  other  direction   the   ray  from  the  polarizer  is  divided 
into  two  constituents  vibrating  in  planes  at  right  angles  to  each 
other,  and  one  of  these  will  traverse  the  analyzer,  hence  such  crys- 
tals will  appear  as  if  self-luminous  in  a  dark  field.     The  experiment 
with  these  crystals  from  the  frog  succeeds  well  with  a  2  mm.  homo- 
geneous immersion. 

As  a  further  illustration  of  anisotropic  objects,  mount  some 
cotton  fibers  in  balsam  (Ch.  IX),  also  some  of  the  lens  paper 
($  125).  These  furnish  excellent  examples  of  vegetable  fibers; 
Striated  muscle  fibers  are  also  very  well  adapted  for  polarizing 
objects. 

(C)  Plcochroism. — This  is  the  exhibition   of  different   tints   as 
the  analyzer  is  rotated.     An  excellent  subject  for  this  will  be  found 
in  blood  crystals. 

§  249.  Starch.  — One  of  the  important  uses  of  a  polariscope  is  for 
the  study  of  starch.  Starch  gives  a  characteristic  black  cross  which 
rotates  as  the  analyzer  is  rotated.  Make  a  thin  slice  of  fresh  raw 

*The  optic  axis  of  doubly  refracting  crystals  is  the  axis  along  which  the 
crystal  is  not  doubly  refracting,  but  isotropic  like  glass.  When  there  is  but 
one  such  axis,  the  crystal  is  said  to  be  uniaxial,  if  there  are  two  such  axes  the 
crystal  is  said  to  be  bi-axial. 

The  crystals  of  carbonate  of  lime  from  the  frog  (see  §164)  are  uniaxial 
crystals.  Borax  crystals  are  bi-axial. 


iy6  MICRO-SPECTROSCOPE  AND  POLARISCOPE     [CH.    17 

potato  with  a  razor  or  other  sharp  knife  and  mount  it  in  water. 
Use  first  a  i6mm.  and  then  a  higher  power.  The  starch  grains 
many  of  them  will  be  found  in  the  potato  'cells.  They  have  the 
general  appearance  of  a  clam  shell.  The  black  cross  is  strikingly 
exhibited  by  the  polariscope.  Starch  grains  of  other  plants  show 
the  same,  but  the  grains  are  smaller  generally  and  therefore  do  not 
bring  out  the  structural  fearures  so  clearly. 

§  250.  Production  of  Colors. — For  the  production  of  gor- 
geous colors,  a  selenite  plate  is  placed  anywhere  between  the  polar- 
izer and  the  analyzer.  If  properly  mounted  the  selenite  is  very 
conveniently  placed  on  the  diaphragm  carrier  ot  the  Abbe  illumin- 
ator, just  above  the  polarizer  ;  an  unmounted  selenite  may  be  placed 
over  the  ocular.  A  thin  plate  or  film  of  mica  also  answers  well. 

It  is  not  necessary  to  use  selenite  or  mica  for  the  production  of 
vivid  colors  in  many  objects.  One  of  the  most  beautiful  prepara- 
tions, and  one  of  the  most  instructive  also,  may  be  prepared  as 
follows  :  Heat  some  xylene  balsam  on  a  slide  until  the  xylene  is 
nearly  evaporated.  Add  some  crystals  of  the  medicine,  sulphonal 
and  warm  till  the  sulphonal  is  melted  and  mixes  with  the  balsam. 
While  the  balsam  is  still  melted  put  on  a  cover- glass.  If  one  gets 
perfect  crystals  there  will  be  shown  beautiful  colors  and  the  black 
cross.  (Clark.) 

It  is  very  instructive  and  interesting  to  examine  many  organic 
and  inorganic  substances  with  a  micro  polarizer. 

REFERENCES    TO    THE    POLARISCOPE     AND    TO  THE    USE    OF 
POLARIZED    LIGHT 

Anthony  &  Brackett,  133;  Behrens;  Behrens,  Kossel  und 
Schiefferdecker;  Carnoy,  61;  Carpenter-Dallinger,  317,  1097;  Clark; 
Daniel,  494;  Davis;  v.  Ebener,  Gamgee;  Halliburton,  36,272;  Hogg, 
133,729;  L/ehmann;  M'Kendrick;.  Na'geli  und  Schwendener,  299; 
Quekett;  Suffolk,  125;  Valentin;  Physical  Review,  I.,  p.  127. 
Daniell,  Physics  for  Medical  Students.  Nichols,  Physics. 

MICRO-CHEMISTRY 

§  251.  During  the  last  decade  the  microscope  has  become  one 
of  the  necessities  of  the  expert  chemist,  and  the  signs  of  the  times 


CI1.    VI}  MICRO-CHEMISTRY  177 

indicate  that  in  every  research  laboratory  of  chemistry  the  micro- 
scope will  become  as  familiar  as  it  now  is  in  research  laboratories  of 
biology.  Its  proper  place  in  chemistry  has  been  admirably  stated 
by  Chamot: 

"  It  is  rather  remarkable  how  slow  American  chemists  have  been  in  re- 
alizing the  importance  of  the  microscope  as  an  adjunct  to  every  chemical 
laboratory.  This  is,  perhaps,  largely  due  to  the  fact  that  few  of  our  students 
in  chemistry  become  familiar  with  the  construction  and  manipulation  of  this 
instrument,  just  as  few  of  them  become  sufficiently  familiar  with  the  spectro- 
scope and  its  manifold  uses;  and  doubtless  also  because  of  the  prevailing  im- 
pression that  a  microscope  is  primarily  an  instrument  for  the  biologist  and  is 
of  necessity  a  most  expensive  luxury.  The  fact  is,  however,  that  this  instru- 
ment is  now  far  from  being  a  luxury  to  the  chemist,  and  the  time  is  not  far 
distant  when  it  will  be  conceded  to  be  as  much  a  necessity  in  every  analytical 
laboratory  as  is  the  balance. 

"  Nor  is  the  apprenticeship  to  its  use  in  chemical  work  long  or  intricate. 

"  Micro-chemical  analysis  should  appeal  to  every  chemist  because  of  its 
neatness,  wonderful  delicacy,  in  which  it  is  not  excelled  even  by  the  spectro- 
scope, and  the  expedition  with  which  an  analysis  can  be  made.  A  complete 
analysis,  intricate  though  it  may  be,  is  a  matter  of  a  few  minutes  rather  than 
of  a  few  hours. 

"  While  there  is  no  good  reason  to  believe,  as  do  some  enthusiasts,  that 
this  new  system  is  to  displace  the  old  analysis  in  the  wet  way,  every  chemist 
should,  nevertheless,  familiarize  himself  with  the  microscope,  its  accessories, 
and  the  elegant  and  time-saving  methods  of  micro-analysis,  thus  enabling  him 
to  examine  qualitatively  the  most  minute  amounts  of  material  with  a  rapidity 
and  accuracy  which  is  truly  marvelous;  not  to  speak  of  the  many  substances 
for  which  no  other  method  of  identification  is  known. 

"  At  present  the  greatest  bar  to  its  general  use  is  the  absence  of  any  well 
defined  scheme,  and  the  absolute  necessity  of  being  well  grounded  in  general 
chemistry.  There  are  no  tables  which  can  be  followed  in  a  mechanical  way  by 
the  student,  but  on  the'  contrary  he  is  obliged  to  exercise  his  knowledge  and 
judgment  at  every  step.  For  this  very  reason  the  introduction  of  this  subject 
into  the  list  of  those  now  taught  is  greatly  to  be  desired." 

The  microscope  is  used  by  the  chemist  to  follow  reactions  in 
minute  quantities  of  material.  This  is  done  by  examining  the 
crystals  which  separate  on  the  addition  of  a  drop  of  reagent  to  a 
drop  of  solution  containing  the  unknown  substance. 

§  252.  Experiment. — To  a  drop  of  distilled  water  on  the 
corner  of  a  slide  add  a  piece  of  calcium  chlorid  about  half  a  milli- 
meter in  diameter.  When  it  is  dissolved  place  a  minute  drop  of 
dilute  sulphuric  acid  (about  ro%)  near  the  drop  of  solution.  With 


i78 


MICRO-CHEMISTRY  [  CH.    VI 


Fig.  140.     Chamot  Chemical  Microscope  (Bausch  &  Lomb  Opt.  Co.). 


CH.    //]  MICRO-CHEMISTRY  179 

a  fine  glass  rod  push  the  two  drops  together.  Shortly  bundles  of 
needle-like  crystals  of  CaSo4-2H2O  will  appear.  This  is  characteris- 
tic of  calcium. 

Lead  nitrate,  strontium  or  barium  chloride  treated  in  the  same 
way  will  yield  fine  amorphous  precipitates.  The  lead  sulphate  will, 
however,  slowly  recrystallize  in  characteristic  forms. 

For  this  examination  a  16  mm.  objective  and  low  ocular  should 
be  employed.  No  cover  glass  is  used. 

§  253.  Slides  for  Microchemistry  and  their  Preparation. — 
These  are  the  regular  1X3  in.  slides  cut  in  half.  The  work  is  done 
on  one  corner  to  avoid  breaking  when  the  slide  is  heated.  It  is 
very  important  to  have  the  slides  clean.  The  slides  are  prepared 
by  leaving  them  over  night  in  cleaning  mixture  (Ch  IX),  and  then 
rinsing  very  thoroughly  in  distilled  water.  The  slides  are  then  left 
in  distilled  water  until  ready  for  use.  They  are  then  wiped  with  a 
clean  glass-towel  or  with  a  double  thickness  of  gauze.  During  the 
whole  process  the  end  of  the  slide  to  be  used  must  not  be  touched 
by  the  fingers.  A  drop  of  water  placed  on  the  slide  should  flatten 
out  and  flow  evenly  over  the  surface.  If  it  heaps  up  in  a  round 
mass  the  slide  is  not  clean. 

\  254.  The  Micro-chemist  should  be  familiar  with  the  appearance  of  the 
different  crystal  forms  under  the  microscope.  He  should  be  especially  familiar 
with  the  appearance  of  crystals  of  the  chlorids,  nitrates,  and  sulfates  of  Sodium, 
Potassium,  and  ammonium;  since  some  of  these  salts  are  sure  to  appear  in 
almost  every  test  drop  examined.  The  following  list  of  substances  have  been 
suggested  by  Dr.  Chamot  as  giving  definite  and  easily  obtained. results.  To 
obtain  good  crystals  dissolve  a  fragment  of  the  substance  in  a  small  drop  of 
water  or  other  solvent  and  let  it  evaporate  spontaneously  until  crystals  appear. 
It  is  better  to  make  the  microscopic  examination  before  the  drying  is  complete. 
Do  not  use  a  cover-glass.  If  one  does  not  obtain  good  crystals,  "seed"  the 
solution  with  some  of  the  crust  which  forms  at  the  edge  of  the  drop  by  push- 
ing some  of  the  crust  into  the  middle  of  the  drop.  This  usually  starts  the 
crystallization. 

Frequently  a  chemically  pure  salt  cannot  be  made  to  yield  satisfactory 
crystals  on  the  evaporation  of  its  solution,  but  beautifully  formed  crystals  will 
result  when  in  the  presence  of  other  compounds.  A  striking  example  is  found 
in  Ammonium  chlorid.  This  salt  fails  to  yield  other  than  dendritic  masses 
when  preparations  are  made  from  the  pure  salt,  but  if  formed  by  metathesis 
and  especially  if  in  the  presence  of  a  difficultly  crystallizable  salt,  well  formed 
isometric  crystals  (cubes)  are  seen. 


i8o  MICRO-CHEMISTRY  [  CH.    \'I 

EXAMPLES    ILLUSTRATING   THE    CRYSTAL   SYSTEMS 

"  Isometric. 

Sodium  chlorid,  potassium  chlorid  potassium  iodid.  Strontium 
nitrate.  Barium  nitrate.  Lead  nitrate.  Potassium  bromid.  Sodium 
bromid. 

Alums  crystallize  in  octahedra,  cubes  or  combinations  of  the  two.  It 
is  well  to  recall  that  the  alums  have  the  general  formula,  M2(RO4):..N2RO4> 
24  H2O,  where  M-  can  be  Al,  Cr,  Mn,  Fe,  In,  Ga,  Tl,  R;  -N-  Na,  K,  Rb, 
Cs,  NH4  Ag,  or  Tl  and  -R-  S  or  Se.  All  alums  are  isomorphous. 

Tetragonal. 

Potassium  copper  chlorid.     Ammonium  copper  chlorid.     Urea. 
Nickel  sulfate  6H2O.     This  salt  is  dimorphic,  crystallizing  also  in  the 
monoclinic  system.     Nickel  sulfate  7H.,O  is  orthorhombic. 

Orthorhombic. 

Asparagin.     Picric  acid.     Acetanilid.     Resorcin. 

Mercuric  chlorid.  Silver  nitrate.  Potassium  sulfate.  Potassium 
nitrate. 

Magnesium  sulfate  7H.2O.  Potassium  chromate.  Sodium  nitrate  (also 
Hexagonal). 

Monoclinic. 

Lactose.  Napthalene.  Potassium  ferric  oxalate.  Sodium  ferric 
oxalate. 

Potassium  chlorate  (sodium  chlorate  is  Isomet.   or  Tetrag. ) 

Lead  acetate.     Copper  acetate  H2O.     Oxalic  acid. 

Ferrous  sulfate,  this  salt  forms  normally  with  7  H2O  and  is  then  Mono- 
clinic,  but  in  presence  of  zinc  sulfate  becomes  Orthorhombic,  and  in 
presence  of  copper  sulfate,  triclinic.  Sodium  Sulfate  lotLO.  Borax. 
Potassium  ferricyanid. 

Triclinic. 

Copper  sulfate  5H2O.     Boric  acid.     Potassium  dichromate. 

Hexagonal. 

Lead  iodid   (according  to   Behrens  PbI2  is  probably   orthorhombic). 
Sodium  nitrate  (also  Orthorhombic).     Bromoform.     lodoform. 

AN   EXERCISE   FOR    PRACTICE 

"  Take  a  fragment  of  ammonium  chlorid,  dissolve  in  a  tiny  drop  of  water 
on  a  slide  and  try  to  obtain  distinct  well  formed  crystals.  Neither  slow  nor 
rapid  evaporation  nor  recrystallization  by  breathing  on  the  preparation  will 
yield  satisfactory  crystals." 

Place  a  small  drop  of  water  on  a  glass  slide,  add  Ferric  chlorid  until  the 
drop  is  distinctly  yellow.  Stir.  At  the  center  of  the  drop  add  two  or  three 
tiny  fragments  of  Ammonium  acetate.  The  preparation  must  not  be  warmed- 


CH.    VI]  MICRO-CHEMISTRY  181 

There  is  formed  Ferric  acetate,  Ammonium  chloric!  and  double  chlorids  of 
ammonium  and  iron.  Study  the  preparation  and  observe  the  following  points, 
i.  Tendency  toward  formation  of  double  salt.  2.  That  the  type  crystal  of 
NH,C1  is  a  cube.  3.  Cubes  may  so  grow  as  to  present  the  appearance  of  a 
rectangular  prism.  4.  In  certain  positions  cubes  have  the  appearance  of  a 
pyramid.  5.  In  other  positions  they  exhibit  a  hexagonal  outline,  thus  simu- 
lating a  polyhedron  of  many  faces.  6.  There  is  scarcely  any  tendency  in 
this  case  toward  the  formation  of  the  dendritic  masses  observed  in  the  first 
experiment.  7.  The  crystals  often  develop  fastest  along  the  diagonal  planes 
so  that  the  regular  faces  are  replaced  by  pyramidal  depressions." 


FIG.  141.  Czapski's  Ocular  Iris-diaphragm  with  cross 
hairs  for  examining  and  accurately  determining  the  axial 
images  of  small  crystals.  The  iris  diaphragm  enables  the 
observer  to  make,  the  field  as  large  or  small  as  desired. 

A.  Longitudinal  Section. 

B.  Transection,  showing  the  cross  lines  and  the  iris 
diaphragm  rcifh  the  projecting  part  at  the  left,  by   which 
the  diaphragm  is  opened  and  closed.      (Zeiss'  Catalog. )  B 


For  directions  and  hints  in  micro-chemical  work  and  crystallography, 
consult  the  various  volumes  of  the  Journal  of  the  Roy.  Micr.  Soc. ;  Zeitschrift 
fur  physiologische  Chemie,  and  other  chemical  journals;  Wormly;  Kletnent 
&  Renard;  Carpenter-Dallinger;  Hogg;  Behrens,  Kossel  und  Schiefferdecker; 
Frey;  Dana,  and  other  works  on  mineralogy;  Davis,  Behrens,  T.  H. — Anleitung 
zur  rnicro-chemischen  Analyse  der  wichtigsten  organischen  Verbindungen. 
Hamburg,  1895-1897.  Microchemische  Technik,  2d  edition*  Hamburg,  1900. 
A  manual  of  michrochemical  analysis  with  an  introductory  chapter  by  J.  W. 
Judd,  London.  1894. 

Especial  attention  is  also  called  to  the  articles  of  Dr.  E.  M.  Chamot 
in  the  Journal  of  Applied  Microscopy  beginning  with  vol  ii.  p.  502,  and  contin- 
ued in  vol  iii.  and  iv. 

TEXTILE    FIBERS,    FOOD    AND    PHARMACOLOGICAL    PRODUCTS 

\  255.  Textile  Fibers. — The  microscope  is  coming  more  and  more  into 
use  for  the  determination  of  the  character  of  textile  fibers,  both  in  the  raw  state 
and  after  manufacture.  As  the  textile  fibers  have  distinctive  characters  it  is 
not  difficult  to  determine  mixtures  in  fabrics  of  various  kinds.  The  student  is 
advised  to  study  carefully  known  fibers,  as  of  cotton,  wool,  linen,  silk,  jute 
etc.,  so  that  he  is  certain  of  the  appearances,  and  then  to  determine  of  what 
fibers  different  fabrics  are  composed.  He  will  be  astonished  at  the  amount  of 
<(  Alabama  wool"  in  supposedly  all  wool  goods. 


182 


FOOD  AND  DRUGS 


[C//.    VI 


\  256.  Food  and  Drugs. — From  the  nature  of  food  and  pharmacological 
products  adulterations  are  in  many  cases  most  accurately  and  easily  determined 
by  microscopic  examination.  The  student  will  find  constant  reference  to  the 
microscopical  characters  of  the  genuine  and  spurious  substances  in  medicines 
and  other  pharmacological  products  in  works  on  pharmacy  or  pharmacology; 
also  in  pharmacological  journals  and  in  druggists  reports. 

For  works  and  articles  upon  textile  fibers  see:  Herzfeld,  J.  Translated  by 
Salter.  The  technical  testing  of  yarns  and  textile  fabrics  with  reference  to 
official  specifications.  London,  1898.  E.  A.  Posselt — The  structure  of  fibers 


FIG.  142.  Ncbelthau's  Traversing  Microscope.  This  instrument  makes 
it  possible  to  go  over  carefully  very  large  objects,  entire  brain  sections,  or  to 
scrutinize  carefully  a  large  amount  of  a  substance  as  in  examinations  for 
adulterations  of  foods  or  drugs.  (From  Leitz'  Catalog. ) 


CH.  VI}  MICRO-METALLOGRAPHY  183 

yarns  and  fabrics.  Philadelphia  and  London,  1891.  Dr.  C.  Rougher — Des 
filements  vegetaux  employes  dans  1'industrie.  Paris,  1873.  Wm.  P.  Wilson 
and  E.  Fahring— ,  The  conditioning  of  wool  and  other  fabrics  in  the  techno- 
logical laboratories  of  the  Philadelphia  Commercial  Museum.  Journal  of  Ap- 
plied Microscopy,  Vol.  II,  (1899)  pp.  290-292,  457-460.  Bulletin  of  the 
National  Association  of  Wool  Growers,  1875,  p.  470.  Proceedings  of  the  Amer- 
Micr.  Soc.,  1884,  pp.  65-68.  Hanausek  and  Winton.  The  Microscopy  of 
Technical  Products;  Winslow,  Elements  of  Applied  Microscopy,  excellent  on 
foods,  drugs,  textile  fibers,  paper.  Besides  these  references  one  is  liable  to 
find  pictures  and  discussions  of  various  fibers  in  general  works  on  the 
microscope,  and  in  technical  and  general  cyclopaedias. 

The  microscopical  Journals  also  contain  occasional  articles  bearing  upon 
this  subject.  See  also  Food  Products  in  bulletins  of  theU.  S.  Dep't  Agr.  Mace, 
E. — Les substances alitnentaire,  etc.,  Paris,  1891.  Schimper,  A.  F.  W.  Anleit. 
ung,  etc.  Jena,  1900.  HughGalt, — The  Microscopy  of  the  starches,  illustrated 
by  photo-micrographs,  London,  1900.  Winton  and  Moeller,  the  Microscopy 
of  Vegetable  Foods.  Greenish,  Micr.  Ex.  Food  and  Drugs.;  Wiley,  Foods 
and  their  Adulterations.  (See  also  the  other  works  in  the  Bibliography  at  the 
end.) 

THE    MICROSCOPE    IN    METALLOGRAPHY 

\  257.  In  the  modern  investigation  of  metals  and  alloys  much  light  has 
been  thrown  upon  the  structural  peculiarities  which  render  some  mixtures 
satisfactory  and  others  unsatisfactory.  There  are  two  great  methods:  First, 
that  of  studying  fractured  surfaces  without  recourse  to  any  reagents.  Second, 
to  polish  a  metallic  surface  carefully  with  emery  or  carborundum  and  finally 
with  rouge  or  diamantine  and  then  etch  it  with  some  acid  for  a  longer  or 
shorter  time.  For  either  method  reflected  light  must  be  used.  For  low  powers 
that  obtained  at  a  good  window  or  by  a  lamp  or  a  lamp  and  bulls  eye  are  good. 
The  illuminating  objectives  ($  31),  i.  e.  objectives  in  which  a  prism  or  reflector 
in  the  objective  reflects  light  down  through  the  lenses  which  act  as  a  conden- 
ser, are  preferable  for  most  work  and  indeed  necessary  if  one  uses  high  powers. 

Elaborate  arrangements  have  been  devised  for  holding  the  piece  of  metal 
on  the  stage,  but  some  beeswax,  or  some  clay  made  plastic  with  glycerin 
answers  well.  For  pictures  of  the  appearances  seen  in  studying  metallic 
surfaces,  see  the  journals  of  engineering  and  metallurgy,  especially  the 
Metallographist,  a  quarterly  publication  devoted  to  the  study  of  metals  with 
special  reference  to  their  physics  and  micro-structure,  etc.  In  twenty-five  or 
more  of  the  great  metal  manufacturing  establishments  special  laboratories  for 
microscopic  examination  and  investigation  have  been  established.  This  is  an 
illustration  of  what  has  frequently  occured — great  manufacturing  interests 
have  outrun  the  universities  in  the  appreciation  and  application  of  methods  of 
research.  Fortunately,  however,  laboratories  are  already  springing  up  in 
connection  with  the  universities,  and  probably  within  a  few  years  every  great 
technical  school  will  have  its  laboratory  of  micro-metallography  where  students 


1 84 


MICRO- ME  TA  L  L  OCR  API!  Y 


\_CH.    VI 


will  have  opportunity  to  perfect  themselves  in  the  preparation,  photography 
and  microscopic  study  of  the  metals  and  alloys. 

Beside  the  sources  of  information  given  above,  see  Dr.  H.  Ost  und  Dt. 
Fr.  Kolbeck,  L,ehrbuch  derchemischen  Technologic  miteinemSchlussabschnitt 
"  Metallurgie."  Hannover,  1901.  Behrens,  T.  H. — Das  mikroskopische 
Gefiige  der  Metalle,  etc.  Hamburg,  1894.  For  an  excellent  bibliography  of 
188  titles;  see  the  Metallographist,  Vol.  I,  1898,  and  appended  to  the  special 
papers  in  all  the  volumes.  Also  in  Iron  Age,  Jan.  27,  1898.  Carpenter-Dal- 
linger,  p.  264;  and  every  number  of  the  Journal  of  the  Royal  Microscopical 
Society  and  Zeit  wiss  Mikroskopie. 


143  144 

FIG.  143.  Zeiss'  Illuminating  Objective.  Light  at  right  angles  to  the  a.vis 
of  the  microscope  is  reflected  by  a  prism  doicn  through  the  lenses  of  the  objec- 
tive upon  the  obj.cct.  This  lights  the  object,  and  rays  from  it  pass  up  through 
the  objective  to  form  the  image  (Zeiss'  Catalog}. 

FIG.  144.  Leitz'  Illuminating  Objective.  The  general  principle  is  the 
same  as  for  Fig.  /^rj. 


CHAPTER  VII 

THE  ABBE  TEST  PLATE  AND  APERTOMETER  ;    EQUIVA- 
LENT   FOCUS   OF    OBJECTIVES    AND  OCULARS; 
CLASS  DEMONSTRATION  IN  HISTOLOGY 
AND  EMBRYOLOGY 

APPARATUS    AND    MATERIAL    FOR    THIS    CHAPTER 

Abbe  test-plate  (?  258);  Apertometer  ($  259);  Tester  for  immersion  liquid 
(I  2t>o);  Microscope  with  250  mm.  tube  and  objectives  (£  262);  Stage  microme- 
ter (  '',.  262);  Filar  micrometer  with  positive  ocular  (£  262);  Oculars  ($264). 

Demonstration  microscopes  and  dissecting  microscope  (Figs.  147-149); 
Traveling  microscope  (Figs.  150-151);  Indicator  or  pointer  oculars  (Figs.  152- 
154);  Compound  microscope  (Fig.  155);  Projection  microscope  (Figs.  158-160). 

TEST   PLATE   AND   APERTOMETER 

'i  258.  On  the  Method  of  Using  Abbe's  Test-Plate. — This  test-plate  is 
intended  for  the  examination  of  objectives  with  reference  to  their  corrections 
for  spherical  and  chromatic  aberration  and  for  estimating  the  thickness  of  the 
cover-glass  for  which  the  spherical  aberration  is  best  corrected. 

"The  test-plate  consists  of  a  series  of  cover-glasses  ranging  in  thickness 
from  0.09  mm.  to  0.24  mm.,  silvered  on  the  under  surface  and  cemented  side 
by  side  on  a  slide.  The  thickness  of  each  is  written  on  the  silver  film.  Groups 
of  parallel  lines  are  cut  through  the  film  and  these  are  so  coarsely  ruled  that 
they  are  easily  resolved  by  the  lowest  powers,  yet  from  the  extreme  thinness 
of  the  silver  they  form  a  very  delicate  test  for  objectives  of  even  the  highest 
power  and  widest  aperture.  To  examine  an  objective  of  large  aperture  the 
plates  are  to  be  focused  in  succession  observing  each  time  the  quality  of  the 
image  in  the  center  of  the  field  and  the  variation  produced  by  using  alter- 
nately central  and  very  oblique  illumination.  When  the  objective  is  perfectly 
corrected  for  spherical  aberration  for  the  particular  thickness  of  cover-glass 
under  examination,  the  contour  of  the  lines  in  the  center  of  the  field  will  be 
perfectly  sharp  by  oblique  illumination  without  any  nebulous  doubling  or 
indistinctness  of  the  minute  irregularities  of  the  edges.  If  after  exactly 
adjusting  the  objective  for  oblique  light,  central  illumination  is  used  no  alter- 
ation of  the  adjustment  should  be  necessary  to  show  the  contours  with  equal 
sharpness." 


i86        •  TEST  PLATE  AND  APERTOMETER  [<f//.    VII 

"  If  an  objective  fulfills  these  conditions  with  any  one  of  the  plates  it  is 
free  from  spherical  aberration  when  used  with  cover-glasses  of  that  thickness; 
on  the  other  hand  if  every  plate  shows  nebulous  doubling  or  an  indistinct 
appearance  of  the  edges  of  the  silver  lines,  with  oblique  illumination,  or  if  the 
objective  requires  a  different  adjustment  to  get  equal  sharpness  with  central 
as  with  oblique  light,  then  the  spherical  correction  is  more  or  less  imperfect." 

"  Nebulous  doubling  with  oblique  illumination  indicates  overcorrection  of 
the  marginal  zone,  want  of  the  edges  without  marked  nebulosity  indicates 
undercorrection  of  this  zone;  an  alteration  of  the  adjustment  for  oblique  and 
central  illumination,  that  is,  a  difference  of  plane  between  the  image  in  the 
peripheral  and  central  portions  of  the  objective  points  to  an  absence  of  con- 
current action  of  the  separate  zones,  which  may  be  due  to  either  an  average 
under  or  overcorrection  or  to  irregularity  in  the  convergence  of  the  rays." 

"  The  test  of  chromatic  correction  is  based  on  the  character  of  the  color 
bands,  which  are  visible  by  oblique  illumination.  With  good  correction  the 
edges  of  the  silver  lines  in  the  center  of  the  field  should  show  but  narrow 
color  bands  in  the  complementary  colors  of  the  secondary  spectrum,  namely, 
on  one  side  yellow-green  to  apple-green  on  the  other  violet  to  rose.  The  more 
perfect  the  correction  of  the  spherical  aberration  the  clearer  this  color  band 
appears." 

"To  obtain  obliquity  of  illumination  extending  to  the  marginal  zone  of 
the  objective  and  a  rapid  interchange  from  oblique  to  central  light  Abbe's 
illuminating  apparatus  is  very  efficient,  as  it  is  only  necessary  to  move  the 
diaphragm  in  use  nearer  to  or  further  from  the  axis  by  the  rack  and  pinion 
provided  for  the  purpose.  For  the  examination  of  immersion  objectives, 
whose  aperture  as  a  rule  is  greater  than  180°  in  air  and  those  homogeneous 
immersion  objectives,  which  considerably  exceed  this,  it  will  be  necessary  to 
bring  the  under  surface  of  the  Test-plate  into  contact  with  the  upper  lens  of 
the  illuminator  by  means  of  a  drop  of  water,  glycerin  or  oil." 

"  In  this  case  the  change  from  central  to  oblique  light  may  be  easily 
effected  by  the  ordinary  concave  mirror  but  with  immersion  lenses  of  large 
aperture  it  is  impossible  to  reach  the  marginal  zone  by  this  method,  and  the 
best  effect  has  to  be  searched  for  after  each  alteration  of  the  direction  of  the 
mirror." 

"  For  the  examination  of  objectives  of  smaller  aperture  (less  than  4o°-5o°) 
we  may  obtain  all  the  necessary  data  for  the  estimation  of  the  spherical  and 
chromatic  corrections  by  placing  the  concave  mirror  so  far  laterally,  that  its 
edge  is  nearly  in  the  line  of  the  optic  axis  the  incident  cone  of  rays  then  only 
filling  one-half  of  the  aperture  of  the  objective.  The  sharpness  of  the  contours 
and  the  character  of  the  color  bands  can  be  easily  estimated.  Differences  in 
the  thickness  of  the  cover-glass  within  the  ordinary  limits  are  scarcely  notice- 
able with  such  objectives." 

"  It  is  of  fundamental  importance  in  employing  the  test  as  above  described 
to  have  brilliant  illumination  and  to  use  an  eye-piece  of  high  power." 

"  When  from  practice  the  eye  has  learnt  to  recognize  the  finer  differences 
in  the  quality  of  the  contour  images  this  method  of  investigation  gives  very 


CH. 


TEST  PLATE  AND  APERTOMETER 


187 


trustworthy  results.     Differences  in  the  thickness  of  cover-glasses  of  o.oi   or 
0.02  mm.  can  be  recognix.ed  with  objectives  of  2  or  3  mm.  focus. 

"  With  oblique  illumination  the  light  must  always  be    thrown  perpendic- 
ularly to  the  direction  of  the  lines." 


FIG.  145.      The   Abbe  Test  Plate,  lines  covered  by  cover-glasses  ranging  in 
thickness  from  0.09  to  0.24  mm. 

"The  quality  of  the  image  outside  the  axis  is  not  dependent  on  spherical 
and  chromatic  correction  in  the  strict  sense  of  the  term.  Indistinctness  of  the 
contours  toward  the  borders  of  the  field  of  view  arises  as  a  rule,  from  unequal 
magnification  of  the  different  zones  of  the  objective;  color  bands  in  the 
peripheral  portion  (with  good  color  correction  in  the  middle)  are  caused  by 
unequal  magnification  of  the  different  colored  images." 

"  Imperfections  of  this  kind,  improperly  called  "  curvature  of  the  field," 
are  shown  to  a  greater  or  less  extent  in  the  best  objectives,  where  the  aperture 
is  considerable." 


FIG.  146.     Abbe  Apertometer. 

\  259.  Determination  of  the  Aperture  of  Objectives  with  an  Apertometer. — 
Excellent  directions  for  using  the  Abbe  Apertometer  may  be  found  in  the  Jour. 
Roy.  Micr.  Soc.,  1878,  p.  19,  and  1880,  p.  20;  in  Dippel,  Zimmerman,  Czapski 
and  Spitta,  Ch.  XIV.  The  following  directions  are  but  slightly  modified  from 
Carpenter-Dallinger,  pp.  394-396.  The  Abbe  apertometer  involves  the  same 
principle  as  that  of  Tolles,  but  it  is  carried  out  in  a  simpler  manner;  it  is 
shown  in  Fig.  146.  As  seen  by  this  figure  it  consists  of  a  semi-circular  plate 
of  glass.  Along  the  straight  edge  or  chord  the  glass  is  beveled  at  45°,  and 
near  this  straight  edge  is  a  small,  perforated  circle,  the  perforation  being  in 


188  TEST  PLATE  AND   APERTOMETER  [CH.    VII 

the  center  of  the  circle.  To  use  the  apertometer  the  microscope  is  placed  in 
a  vertical  position,  and  the  perforated  circle  is  put  under  the  microscope  and 
accurately  focused.  The  circular  edge  of  the  apertometer  is  turned  toward  a 
window  or  plenty  of  artificial  light  so  that  the  whole  edge  is  lighted.  When 
the  objective  is  carefully  focused  on  the  perforated  circle  the  draw-tube  is 
removed  and  in  its  lower  end  is  inserted  the  special  objective  which  accom- 
panies the  apertometer.  This  objective  and  the  ocular  form  a  low  power  com- 
pound microscope,  and  with  it  the  back  lens  of  the  objective,  whose  aperture 
is  to  be  measured,  is  observed.  The  draw-tube  is  inserted  and  lowered  until 
the  back  lens  of  the  objective  is  in  focus.  "In  the  image  of  the  back  lens 
will  be  seen  stretched  across,  as  it  were,  the  image  of  the  circular  part  of  the 
apertometer.  It  will  appear  as  a  bright  band,  because  the  light  which' enters 
normally  at  the  surface  is  reflected  by  the  bevel  part  of  the  chord  in  a  vertical 
direction  so  that  in  reality  a  fan  of  180°  in  air  is  formed.  There  are  two  sliding 
screens  seen  on  either  side  of  the  apertometer;  they  slide  on  the  vertical  circu- 
lar portion  of  the  instrument.  The  images  of  these  screens  can  be  seen  in  the 
image  of  the  bright  band.  These  screens  should  noiv  be  moved  so  that  their 
edges  j list  totich  the  periphery  of  the  back  lens.  They  act,  as  it  were,  as  a 
diaphragm  to  cut  the  fan  and  reduce  it,  so  that  its  angle  just  equals  the  aperture 
of  the  objective  and  no  more."  "  This  angle  is  now  determined  by  the  arc  of 
glass  between  the  screens;  thus  we  get  an  angle  in  glass  the  exact  equivalent 
of  the  aperture  of  the  objective.  As  the  numerical  apertures  of  these  arcs  are 
engraved  on  the  apertometer  they  can  be  read  off  by  inspection.  Nevertheless 
a  difficulty  is  experienced,  from  the  fact  that  it  is  not  easy  to  determine  the 
exact  point  at  which  the  edge  of  the  screen  touches  the  periphery  of  the  back 
lens,  or  as  we  prefer  to  designate  it,  the  limit  of  aperture,  for  curious  as  the 
expression  may  appear  we  have  found  at  times  that  the  back  lens  of  the  objec- 
tive is  larger  than  the  aperture  of  the  objective  requires.  In  that  case  the 
edges  of  the  screen  refuse  to  touch  the  periphery." 

In  determining  the  aperture  of  homogeneous  immersion  objectives  the 
proper  immersion  fluid  should  be  used  as  in  ordinary  observation.  So,  also, 
with  glycerin  or  water  immersion  objectives. 

\  260.  Testing  Homogeneous  Immersion  Liquid.— In  order  that  one 
may  realize  the  full  benefit  of  the  homogeneous  immersion  principle  it  is 
necessary  that  the  homogeneous  immersion  liquid  shall  be  truly  homogeneous. 
In  order  that  the  ordinary  worker  may  be  able  to  test  the  liquid  used  by  him, 
Professor  Hamilton  L,.  Smith  devised  a  tester  composed  of  a  slip  of  glass  in 
which  was  ground  accurately  a  small  concavity  and  another  perfectly  plain 
slip, to  act  as  cover.  (See  Proc.  Ainer.  Micr.  Soc.;  1885,  p.  83.)  It  is  readily 
seen  that  this  concavity,  if  filled  with  air  or  any  liquid  of  less  refractive  index 
than  glass,  acts  as  a  concave  or  dispersing  lens.  If  filled  with  a  liquid  of 
greater  refractive  index  than  glass,  the  concavity  acts  like  a  convex  lens,  but 
if  filled  with  a  liquid  of  the  same  refractive  index  as  glass,  that  is,  liquid  opti- 
cally homogeneous  with  glass,  then  there  is  no  effect  whatever. 

In  using  this  tester  the  liquid  is  placed  in  the  concavity  and  the  cover  put 
on.  This  is  best  applied  by  sliding  it  over  the  glass  with  the  concavity.  A 
small  amount  of  the  liquid  will  run  between  the  two  slips,  making  optical 


CH.   /'//  |  FOCUS  OF  OBJECTIVES  •      189 

contact  on  both  surfaces.  One  should  be  careful  not  to  include  air  bubbles  in 
the  concavity.  The  surfaces  of  the  glass  are  carefully  wiped  so  that  the  image 
will  not  be  obscured.  An  adapter  with  society  screw  is  put  on  the  microscope 
and  the  objective  is  attached  to  its  lower  end.  In  this  adapter  a  slot  is  cut  out 
of  the  right  width  and  depth  to  receive  the  tester  which  is  just  above  the 
objective.  As  object  it  is  well  to  employ  a  stage  micrometer  and  to  measure 
carefully  the  diameter  of  the  field  without  the  tester,  then  with  the  tester  far 
enough  inserted  to  permit  of  the  passage  of  rays  through  the  glass  but  not 
through  the  concavity,  and  finally  the  concavity  is  brought  directly  over  the 
back  lens  of  the  objective.  This  can  be  easily  determined  by  removing  the 
ocular  and  looking  down  the  tube.  • 

Following  Professor  Smith's  directions  it  is  a  good  plan  to  mark  in  some 
way  the  exact  position  of  the  tube  of  the  microscope  when  the  micrometer  is 
in  focus  without  the  tester,  then  with  the  tester  pushed  in  just  far  enough  to 
allow  the  light  to  pass  through  the  plane  glass  and  finally  when  the  light 
traverses  the  concavity.  The  size  of  the  field  should  be  noted  also  in  the 
three  conditions  (#  57-58). 

It  is  seen  by  glancing  at  the  following  table  that  whenever  the  liquid  in 
the  tester  is  of  lower  index  than  glass,  the  concavity  with  the  liquid  acts 
as  a  concave  lens,  or  in  other  words  like  an  amplifier  (p.  123) ,  and  the  field  is 
smaller  than  when  no  tester  is  used.  It  is  also  seen  that  as  the  liquid  in  the 
concavity  approaches  the  glass  in  refractive  index,  the  field  approaches  the 
size  when  no  tester  is  present.  It  is  also  plainly  shown  by  the  table  that  the 
greater  the  difference  in  refractive  index  of  the  substance  in  the  concavity 
and  the  glass,  the  more  must  the  tube  of  the  microscope  be  raised  to  restore 
tbe  focus. 

If  a  substance  of  greater  refraction  than  glass  is  used  in  the  tester  the 
field  is  larger,  /.  e. ,  the  magnification  less,  and  one  would  have  to  turn  the 
tube  down  instead  of  up  to  restore  the  focus. 

The  table  given  below  indicates  the  changes  when  using  a  tester  prepared 
by  the  Gundlach  Optical  Co. ,  and  used  with  a  16  mm.  apochromatic  objective  of 
Zeiss,  X4  compensation  ocular,  achromatic  condenser,  i.oo  N.  A.  (Fig.  47): 


Tester  and  Liquid  in  the  Concavity 


Size  of  the 
Field 


Elevation  of  the  Tube 

necessary  to 
Restore  the  Focus 


No  tester  used 1.825  mm.__  Standard  position  _. 

Whole  thickness  of  the  tester  at  one  end, 

not  over  the  cavity 1.85  mm. No  change  of  focus. 

Tester  with  water.../. 1.075  mm.__  Tube  raised  3^  mm. 

Tester  with  95%  alcohol 1.15  mm ....     3mm. 

Tester  with  kerosene 14  mm :  ....     2mm. 

Tester  with  Gundlach  Opt.  Go's  horn,  liquid  1.825  mm.  __    ....     T'/5  mm. 

Bausch  &  I/omb  Opt.  Go's  horn,  liquid i  825  mm.__    ....     T25°(j  mm. 

Leitz' horn,  liquid 1.825  mm._.    ....     f^j  mm. 

Zeiss' hom.  liquid 1.825  mm. _.    •     »     •     •     TS°O  mm- 

%  261.     Equivalent    Focus   of  Objectives  and  Oculars. — To   work  out   in 
proper  mathematical  form  or  to  ascertain  experimentally  the  equivalent  foci 


190  FOCUS  OF  OBJECTIVES  [C//.    VII 

of  these  complex  parts  with  real  accuracy  would  require  an  amount  of  knowl- 
edge and  of  apparatus  possessed  only  by  an  optician  or  a  physicist.  The  work 
may  be  done,  however,  with  sufficient  accuracy  to  supply  most  of  the  needs  of 
the  working  microscopist.  The  optical  law  on  which  the  following  is  based 
is  : — "  The  size  of  object  and  image  varies  directly  as  their  distance  from  the 
center  of  the  lens." 

By  referring  to  Figs.  14,  16,  26,  it  will  be  seen  that  this  law  holds  good. 
When  one  considers  compound  lens  systems  the  problem  becomes  involved, 
as  the  center  of  the  lens  system  is  not  easily  ascertainable  hence  it  is  not 
attempted,  and  only  an  approximately  accurate  result  is  sought. 

§  262.  Determination  of  the  Equivalent  Focus  of  Ob- 
jectives.— Look  into  the  objective  to  be  tested  and  locate  the  posi- 
tion of  the  back  lens.  Indicate  this  on  the  outside  of  the  objective 
mount.  This  is  not  usualty  at  the  optical  center,  but  a  near  enough 
approximation  for  this  experiment.  Put  the  objective  in  position 
on  a  microscope  whose  draw-tube  may  be  extended  250  mm.  z.  e.  : 
sufficiently  to  give  a  tube-length  of  250  mm.  If  the  draw- tube  is 
not  of  sufficient  length  put  on  an  extension  piece. 

Select  a  positive  ocular.  One  of  the  Filar  micrometers  is  very 
satisfactory  (Fig.  119).  A  Huygenian  ocular  is  not  satisfactory 
for  this  purpose.  Use  a  stage  micrometer  as  object.  With  exten- 
sion piece  and  draw-tube  make  the  distance  between  the  back  lens 
of  the  objective  and  the  position  of  the  cross  lines  of  the  filar  mi- 
crometer 250  mm.  This  is  so  that  the  image  distance  shall  be 
250  mm. 

Arrange  the  filar  micrometer  so  that  its  movable  line  shall  be 
parallel  with  one  of  the  lines  of  the  stage  micrometer,  and  then  proceed 
to  measure  the  space,  making  several  measurements  and  taking  the 
average  as  directed  in  §  190.  But  in  this  case  it  is  necessary  to 
know  the  size  of  the  real  image  in  millimeters.  The  pitch  of  the 
screw  we  will  suppose  is  ^mm.  as  in  the  one  figured  (Fig.  119) 
then  the  whole  revolution  will  move  the  traversing  line  ^  mm., 
and  the  partial  revolutions  may  be  read  on  the  graduated  drum  each 
graduation  representing  a  movement  of  0.005  mm-  or  5/'-  Suppose 
it  requires  2.50  revolutoins  of  the  drum  to  pass  the  movable  line 
over  y1^  of  a  millimeter  on  the  stage  micrometer.  Then  the  size 
of  the  real  image  of  T^  mm.  is  two  and  one-half  revolutions  multi- 
plied by  the  value  of  one  revolution  or  the  pitch  of  the  screw  which 
is  one-half  of  a  millimeter  thus  :  2.5OX  0.5  =  1.25  mm.  Now  if  the 
object  is  yV  mm.  and  the  real  image  is  1.25  mm.  the  magnifica- 


CH.    VII]  FOCUS  OF  OCULARS  191 

tion  of  the  real  image  is  1.25-7-0.1  =  12.5  or  the  real  image  is  i2l/> 
times  as  large  as  the  object  (Figs.  26,  109.) 

To  find  the  equivalent  focus  of  this  objective  knowing  its 
magnification  at  250  mm.  one  has  simply  to  apply  the  law  as  shown 
graphically  in  Fig.  109,  viz;  The  si/.e  of  object  and  image  are  di- 
rectly as  their  distances  from  the  center  of  the  lens  :  The  distance  of 
the  object  from  tlie  lens  is  with  the  microscope  very  nearly  the 
principal  focal  distance  and  is  designed  by  f.  The  formula  is  then 
written:  the  object  is  to  the  image  as  the  principal  focal  dis- 
tance is  to  the  imagedistance  (25omm.)  or  o:i::  f:25o  mm.  In  this 
case  all  the  factors  are  known  except  f.  Then  1.25:0.  i::f: 
250  whence  f— 20.  Or  as  the  magnification  of  the  real  image  is 
known  to  be  12.5  the  formula  may  read  12.5:1  ::f:25o  whence  f= 
20  as  before.  By  referring  to  figures  109  it  is  seen  that  if  the  sim- 
ple lens  had  a  principal  focal  distance  of  20  mm.  and  the  image  dis- 
tance is  250  mm.  then  the  real  image  is  12.5  times  the  length  of 
the  object,  since  the  distances  from  the  center  of  the  lens  to  the  object 
(20  mm)  and  image  (250  mm.)  are  in  the  proportion  of  i  to  12.5. 

§  263  Determination  of  Initial  or  Independent  Magnifica- 
tion of  the  Objective. — The  Initial  magnification  means  simply 
the  magnification  of  the  real  image  (A'B1,  Fig.  26,  also  Fig  109)  un- 
affected by  the  ocular.  It  may  be  determined  experimentally  exact- 
ly as  described  in  §  262.  For  example,  the  image  of  the  object 
(y-g-  mm.)  measured  by  the  ocular  micrometer,  at  a  distance  of  250 
mm.  is  1.25.,  z'.  <?. ,  it  is  12.5  times  magnified,  hence  the  initial  mag- 
nification of  the  20  trim,  objective  is  12.5. 

Knowing  the  equivalent  focus  of  an  objective,  one  can  deter- 
mine its  initial  magnification  by  dividing  250  mm.  by  the  equivalent 
focus  in  millimeters.  Thus  the  initial  magnification  of  a  5  mm. 
objective  is  ^f-  =  50;  of  a  3  mm.,  -f-  —  83.3;  of  a  2  mm.,  -:]"  =125. 

§  264.  Determining  the  Equivalent  Focus  of  an  Ocular. — 
If  one  knows  the  initial  magnification  of  the  objective  (  §  263)  the 
approximate  equivalent  focus  of  the  ocular  can  be  determined  as 
follows  : 

The  distance  between  the  position  of  the  real  image,  a  position 
indicated  in  the  ocular  by  a  diaphragm,  and  the  back  lens  of  the 
objective  should  be  made  250  mm.,  as  described  in  §  262-263,  then 
by  the  aid  of  Wollaston's  camera  lucida  the  magnification  of  the 


192 


FOCUS  OF  OCULARS 


\CH  VII 


whole  microscope  is  obtained  as  described  in  §  176.  As  the  initial 
power  of  the  objective  is  known,  the  power  of  the  whole  microscope 
must  be  due  to  that  initial  power  multiplied  by  the  power  of  the  oc- 
ular, the  ocular  acting  like  a  simple  microscope  to  magnify  the  real 
image  (Fig  26). 

Suppose  one  has  a  50  mm.  objective;  its  initial  power  will  be 
approximately  5.  If  with  this  objective  and  an  ocular  of  unknown 
equivalent  focus  the  magnification  of  the  whole  microscope  is  50, 
then  the  real  image  or  initial  power  of  the  objective  must  have  been 
multiplied  10  fold.  Now  if  the  ocular  multiplies  the  real  image  10 
fold  it  has  the  same  multiplying  power  as  a  simple  lens  of  25  mm. 
focus,  for,  using  the  same  formula  as  before  :  (o  ;  i  ::f:25o  mm.) 
5 150::  £1250.  Whence  f=25,  the  equivalent  focus  of  the  ocular. 

For  a  discussion  of  the  equivalent  focus  of  compound  lens-systems,  see 
modern  works  on  physics  ;  see  also  C.  R.  Cross,  on  the  Focal  Length  of  Micro- 
scopic Objectives,  Franklin  Institute  Jour;,  1870,  pp.  401-402;  Monthly  Micr. 
Jour.,  1870,  pp.  149-159.  J.  J.  Woodward  on  the  Nomenclature  of  Achromatic 
Objectives,  Amer.  Jour.  Science,  1872,  pp.  406-414;  Monthly  Micr.  Jour., 
1872,  pp.  66-74.  W.  S.  Franklin,  Method  of  determining  focal  lengths  of 
microscope  lenses.  Physical  Review,  Vol.  I,  1893,  p.  142.  See  pp.  1119-1131 
of  Carpenter-Dallinger.  for  mathematical  formulae  ;  also  Daniell,  Physics  for 
medical  students ;  Czapski,  Theorie  der  optischen  Instrumente ;  Dippell, 
Nageli  und  Schwendener,  Zimmermann.  E.  M.  Nelson,  J.  R.  M.  S.  1898,  p. 
362,  1900,  pp.  162-169.  Jour-  Quekett  Micr.  Club,  vol.  V.  pp.  456,  462.  A.  E. 
Wright,  Principles  of  Microscopy,  and  in  Jour.  Roy.  Micr.  Soc. ,  1904,  p.  279; 
Spitta,  Microscopy;  Edser,  Light  for  Students;  Conrad  Beck,  Cantor  Lectures. 


FiG.  147.  Simple  Demonstration  Microscopes.  The  upper  figure  has  a 
kind  of  stage  with  clips  to  hold  the  specimen.  The  lens  may  be  focused  up  and 
down  by  sliding  it  on  the  standard.  For  observation  it  is  held  between  the 
eye  and  the  source  of  light.  In  the  loiver  figure  the  lens  is  supported  by  a 
handle  and  may  be  used  something  as  a  reading  glass.  (From  Leitz' 
Catalog. ) 


CH.    VII}  CLASS  DEMONSTRATIONS 

DEMONSTRATION    MICROSCOPES    AND    INDICATORS 


193 


§  265.  Simple  Microscope. — Holding  the  simple  microscope 
in  one  hand  and  the  specimen  in  the  other,  has  always  been  used 
for  demonstration,  but  for  class  demonstration  it  is  necessary  to 
have  microscope  and  specimen  together  or  the  part  to  be  observed 
by  the  class  is  frequently  missed.  Originally  blocks  of  various 
kinds  to  hold  both  microscope  and  specimen  were  devised,  but  with- 
in the  last  few  years  excellent  pieces  of  apparatus  have  been  devised 
by  several  opticians  for  the  purpose.  The  accompanying  figure 
shows  one  of  the  best  forms. 


FIG.  n>S.  Demonstration  com- 
pound microscope  of  Leilz.  Leitz 
no:c  furnishes  a  fine  adjustment  in 
the  form  of  an  intermediate  piece  be- 
tween the  objective  and  the  tube.  This 
has  in  it  a  screw  ichieh  is  turned  by  a 
milled  ring.  For  the  objectives  em- 
ployed it  makes  an  efficient  fine  ad- 
justment and  renders  it  possible  for 
each  person  to  adjust  the  microscope 
slightly  without  endangering  the  loss 
of  field. 


S  266.  Compound  Demonstration  Microscope. — This  was 
originally  called  a  clinical  or  pocket  microscope.  It  is  thus 
described  by  Mayall  in  his  Canton  Lectures  on  the  history  of  the 
microscope  :  "A  small  microscope  was  devised  by  Tolles  for  clini- 
cal purposes  which  seems  to  me  so  good  in  every  way  that  I  must 
ask  special  attention  for  it.  The  objective  is  screwed  into  a  sliding 


194 


CLASS  DEMONSTRATIONS 


\_CH.   VII 


tube,  and  for  roughly  focusing  the  sliding  motion  suffices  ;  for  fine 
adjustment,  the  sheath  is  made  to  turn  on  a  fine  screw  thread  on  a 
cylindrical  tube,  which  serves  also  as  a  socket  carrier  for  the  stage. 
The  compound  microscope  is  here  reduced  to  the  simplest  form  I 
have  met  with  to  be  a  really  servicable  instrument  for  the  purpose 
in  view;  and  the  mechanism  is  of  thoroughly  substantial  character. 
I  commend  this  model  to  the  notice  of  our  opticians. ' ' 


FIG.   149.     Dissecting  microscope.     This  is  convenient  for  demonstrations 
of  rather  large  objects.     If  they  are  transparent  then  the  mirror  is  used.     If 
the  objects  are  opaque  they  must  be  lighted  by  a  mirror  above  the  stage  or  by  a 
bull's  eye  condenser.     In  this  one  the  focusing  is  done  by  a  rack  and  pinion. 
(Cut  loaned  by  the  Bausch  &  Lomb  Optical  Co.) 

Since  its  introduction  by  Tolles  many  opticians  have  produced 
excellent  demonstration  microscopes  of  this  type,  but  most  of  them 
have  not  preserved  a  special  mechanism  for  fine'  adjustment.  With 
it  one  can  demonstrate  with  an  objective  of  6  mm.  satisfactorily.  It 


C/L    VII]  GLASS  DEMONSTRATIONS 


195 


has  a  lock  so  that  once  the  specimen  is  in  the  right  position  and  the 
instrument  focused  it  may  be  passed  around  the  class.  For  observa- 
tion it  is  only  necessary  for  each  student  to  point  the  microscope 
toward  a  window  or  a  lamp. 


FiG.  150.     Traveling  microscope  sset  up  for  work  (From  Leitz'  Catalog.) 


196 


CLASS  DEMONSTRA  TIONS 


[C7/.   VII 


A  modification  of  this  clinical  microscope  was  made  by  Zent- 
mayer  in  which  the  microscope  was  mounted  on  a  board  and  a  lamp 
for  illuminating  the  object  was  placed  at  the  right  position. 


FIG.  151. 
Catfllog. ) 


Traveling  microscope  folded  up  and  in  its  case.     (From   Leitz 


§  267.  Traveling  Microscope. — For  many  years  the  French 
opticians  have  produced  most  excellent  traveling  microscopes.  The 
opticians  of  other  countries  have  also  brought  out  serviceable  in- 
struments. In  the  one  here  figured  Mr.  Leitz  has  combined  in  an 
admirable  way  a  traveling  microscope  and  a  laboratory  instrument. 
For  the  needs  of  the  pathologist  and  sanitary  inspector  a  microscope 
must  possess  compactness  and  also  the  qualities  which  render 


CH. 


CLASS  DEMONSTRATIONS 


197 


it  usable  for  nearly  all  the  purposes  required  in  a  laboratory. 
This  instrument  is  a  type  of  such  apparatus  which  has  grown  up 
with  the  needs  of  advancing  knowledge. 

£  268.  Indicator  or  Pointer  Ocular. — This  is  an  ocular  in 
which  a  delicate  pointer  of  some  kind  is  placed  at  the  level  where 
the  real  image  of  the  microscope  is  produced.  It  is  placed  at  the 
same  level  as  the  ocular  micrometer,  and  the  pointer  like  the  micro- 
meter is  magnified  with  the  real  image  and  appears  as  a  part  of  the 
projected  image  (Fig.  154).  By  rotating  the  ocular  or  the  pointer 
any  part  of  the  real  image  may  be  pointed  out  as  one  uses  a  pointer 
on  a  wall  or  blackboard  diagram.  By  means  of  the  indicator  eye- 
piece one  can  be  certain  that  the  student  sees  the  desired  object, 
and  is  not  confused  by  the  multitude  of  other  things  present  in  the 
field.  This  device  has  been  invented  many  times.  It  illustrates 


FIG.  152 


FIG.  153 


FIG.  154 


FIG.  152.  Indicator  ocular  ivith  metal  pointer  like  the  one  devised  by 
Quekett  (Leiiz'  catalog). 

FIG.  153.  Indicator  ocular  with  a  fine  hair  from  a  camel's  hair  brush  on 
the  ocular  diaphragm  to  serve  as  a  pointer  (P).  This  projects  about  halfway 
across  the  diaphragm  opening.  On  (he  opposite  side  are  shown  two  rays  from 
the  microscope  to  indicate  that  the  real  image  is  formed  at  the  level  of  the  ocu- 
lar diaphragm. 

FIG.  154.  Field  of  the  microscope  with  a  mammalian  blood  preparation 
to  sho:c  the  use  of  the  indicator  (P)  for  pointing  out  a  white  blood  corpuscle. 


198 


CLASS  DEMONSTRATIONS 


[CH.   VII 


FIG.  155.  Compound  Microscope  with  triple  nose-piece  and  objectives  and 
ocular  in  position.  The  ring  below  the  condenser  is  draivn  to  one  side.  This 
ring  is  for  blue  glass  or  for  a  central-stop  to  use  in  dark-ground  illumination 
(|  /oj).  In  demonstrations  it  is  a  great  advantage  to  have  a  fine  hair  in  the 
ocular  as  shown  in  Figs.  153-154.  (Cut  loaned  by  Williams,  Broivn  &  Earle> 
Phila.} 


CH.    I'll} 


CLASS  DEMONSTRATIONS 


199 


well  the  adage:  "necessity  is  the  mother  of  invention,"  for  what 
teacher  has  not  been  in  despair  many  times  when  trying  to  make  a 
student  see  a  definite  object  and  neglect  the  numerous  other  objects 
in  the  field.  So  far  as  the  writer  has  been  able  to  learn,  Quekett 
was  the  first  to  introduce  an  indicator  ocular  with  a  metal  pointer 
which  was  adjustable  and  could  be  turned  to  any  part  of  the  field 
or  wholly  out  of  the  field.  See  Fig.  152,  §  140. 

It  is  not  known  who  adopted  the  simple  device  of  putting  a 
fine  hair  on  the  diaphragm  of  the  ocular  as  shown  in  Fig.  153.  This 
may  be  done  with  any  ocular,  positive  or  negative.  One  may  use  a 
little  mucilage,  Canada  balsam  or  any  other  cement  to  stick  the 
hair  on  the  upper  face  of  the  diaphragm  so  that  it  projects  about 
half  way  across  the  opening.  When  the  eye-lens  of  the  Huygenian 
ocular  is  screwed  back  in  place  the  hair  should  be  in  focus.  If  it  is 
not  screw  the  eye-lens  out  a  little  and  look  again.  If  it  is  not  now 
sharp,  the  hair  is  a  little  too  high  and  should  be  depressed  a  little. 
If  it  is  less  distinct  on  screwing  out  the  ocular  it  is  too  low  and 
should  be  elevated.  One  can  soon  get  it  in  exact  focus.  Of  course 
it  may  be  removed  at  any  time. 

§  269.  Marking  the  Position  of  Objects. — In  order  that 
one  may  prepare  a  demonstration  easily  and  certainly  in  a  short 
time  the  specimens  to  be  shown  must  be  marked  in  some  way.  An 
efficient  and  simple  method  is  to  put  rings  of  black  or  colored  shellac 
around  the  part  to  be  demonstrated.  For  this  the  Marker,  Figs. 
70-72,  is  employed.  For  temporary  marking  an  ink  line  may  be 
put  on  with  a  pen. 


FIG.  156 


FIG.  157 


FIG.  156.     Ring  around  one  of  the  sections  of  a  series  for  demonstrating 
some  organ  especially  well. 

FIG.  157.     Figure  of  a    microscopic  preparation    with  a  ring  around  a 
small  part  to  show  the  position  of  some  structural  feature. 


CLASS  DEMONSTRATIONS 


[CH.   VII 


THE    PROJECTION    MICROSCOPE 

§  270.  Projection  Microscope.— One  of  the  most  useful  and 
satisfactory  means  at  the  disposal  of  the  teacher  of  Microscopic 
Anatomy  and  Embryology  for  class  demonstrations  is  the  Projection 
Microscope.  With  it  he  can  show  hundreds  of  students  as  well  as 
one,  the  objects  which  come  within  the  range  of  the  instrument. 

It  is  far  more  satisfactory  than  microscopic  demonstrations,  for 
with  the  projection  microscope  the  teacher  can  point  out  on  the 
screen  exactly  the  structural  features  and  organs  'which  he  wishes 
to  demonstrate,  and  he  can  thus  be  certain  that  the  students  know 
exactly  what  is  to  be  studied.  Unless  one  employs  a  pointer  ocular 
(Fig.  153),  there  is  no  certainty  that  the  student  selects  from  the 
multitude  of  things  in  the  microscopic  field  the  one  which  is  meant 
by  the  teacher.  Like  all  other  means,  however,  the  projection 


FIG.  158.  Diagram  of  Adams'  Solar  Microscope.  This  illustrates  well 
•the  advantage  of  some  form  of  projection  microscope  for  demonstration  pur- 
poses. 

a,  b,  c,  d,  <?,  f.  Rays  from  the  sun  striking  the  mirror  A-B,  and  being  re- 
flected horizontally  to  the  condensing  lens  C-D.  The  condensing  lens  concen- 
trates the  light  upon  the  object  E-F.  From  the  object  the  light  passes  to  the 
objective  G-H.  The  objective  then  projects  an  enlarged  image  [-K  upon  the 
screen  at  L-M.  N-O  opening  in  the  shutter. 

The  action  is  exactly  like  that  of  a  magic  lantern  except  that  an  object  is 
used  instead  of  a  lantern  slide,  and  the  objective  gives  a  greater  magnification 
than  the  one  used  on  a  magic  lantern.  (From  George  Adams  Essays.  1787.} 


r//. 


CLASS  DEMONSTRA  TIONS 


201 


microscope  is  limited.  With  it  one  can  show  organs  both  adult  and 
embryonic,  and  the  general  morphology.  For  the  accurate  demon- 
stration of  cells  and  cell  structure  the  microscope  itself  must  be  used. 
As  a  general  statement  concerning  the  use  of  the  projection  micro- 
scope for  demonstration  purposes,  it  may  be  said  that  it  is  entirely 
satisfactory  for  objects  and  details  which  show  under  the  microscope 
with  objectives  up  to  16  mm.  equivalent  focus.  For  objects  and 
details  requiring  objectives  higher  than  16  mm.  focus  in  ordinary 
microscopic  observations,  the  projection  microscope  is  unsatisfactory 
with  large  classes. 


Homo    5 
Slide  10 

See's 

20M 
1901 


FIG.  159.  Slide  of  several  sections  with  a  black  mask.  The  mask  is  per- 
forated over  the  sections  to  be  demonstrated  ivith  the  microscope  or  the  pro- 
jection microscope.  It  is  put  on  the  back  of  the  slide  and  not  on  the  cover- 
glass. 

Unless  one  has  a  mask  something  like  this  the  light  is  so  dazzling  that  it 
is  almost  impossible  to  find  the  proper  sections.  It  is  easily  removed  by  plac- 
ing the  slide  on  :cet  blotting  paper. 


FIG.  1 60.  Projection  Microscope.  This  figure  illustrates  a  modern  pro- 
jection microscope  with  an  arc  light  for  radiant.  Such  a  projection  micro- 
scope is  available  whenever  there  is  an  electric  current;  and  is  of  the  greatest 
use  in  projecting  microscopic  objects  ranging  from  60  mm,  to  ^  of  a  millimeter 
so  that  a  large  class  can  see  all  the  details.  Its  use  in  drawing  was  pointed  out 
in  '',,  209.  (From  Gage,  Origin  and  Development  of  the  Projection  Microscope.) 


202 


CLASS  DEMONSTRATIONS 


\_CH.   VII 


The  microscope  for  the  individual  student  and  the  projection 
microscope  for  the  teacher  furnish  most  efficient  aids  in  getting 
back  to  nature  in  the  study  of  minute  structure  and  morphology. 
Models  and  diagrams  are  very  desirable  aids  in  study  and  teaching, 
but  the  real  things  should  never  be  made  secondary  to  models  and 
diagrams.* 


*  For  a  full  consideration  of  the  Projection  Microscope  see  :  Gage  :  Part 
I.,  The  Origin  and  Development  of  the  Projection  Microscope  ;  Part  II.,  Mod- 
ern Projection  with  directions  for  installation  and  use. 


FIG.  i6oa.      Watson  &  Sons'  Edinburgh  Students'  Microscope  (Stand  G). 


CHAPTER   VIII 


PHOTOGRAPHING   OBJECTS   WITH   A  VERTICAL    CAM- 
ERA; PHOTOGRAPHING  LARGE  TRANSPARENT  OB- 
JECTS; PHOTOGRAPHING  WITH  A  MICROSCOPE 
(A)  TRANSPARENT  OBJECTS;  (B)  OPAQUE 
OBJECTS    AND    THE    SURFACES    OF 
METALS  AND  ALLOYS  ;  ENLARGE- 
MENTS;    LANTERN     SLIDES; 
BACTERIAL       CULTURES. 


APPARATUS    AND    MATERIAL    FOR    THIS    CHAPTER 

Vertical  camera  with  photographic  objectives  (Fig.  161),  small  vertical 
camera  with  special  microscope  stand  for  embryos,  etc.  (Fig.  165);  arrange- 
ment of  camera  for  large  transparent  objects  (Fig.  169),  photo-micrographic 
camera  (Fig.  172);  photographic  objectives  for  gross  and  microscopic  work 
(Figs.  162,  166-168) ;  microscope,  microscopic  objectives  and  projection  oculars 
(Figs.  174-175);  color  screens,  lamps,  dry  plates  and  the  chemicals  and  appara- 
tus necessary  for  developing,  printing,  etc. 

t  271.  Nothing  would  seem  more  natural  than  that  the  camera,  fitted 
with  a  photographic  objective  or  with  a  microscopic  objective,  should  be 
called  into  the  service  of  science  to  delineate  with  all  their  complexity  of 
detail,  the  myriads  of  forms  studied. 

For  photographing  many  objects  in  nature  the  camera  remains  horizontal 
or  approximately  so,  but  for  a  great  many  of  those  studied  in  botany,  zoology, 
mineralogy  and  in  anatomy  the  specimens  cannot  be  safely  or  conveniently 
put  in  the  vertical  position  necessary  for  a  horizontal  camera.  This  difficulty 
has  been  overcome  by  using  a  mirror  or  a  45-degree  prism.  These  are  practi- 
cally alike  and  have  the  defect  of  producing  a  picture  with  the  inversion  of  a 
plane  mirror. 

VERTICAL    CAMERA* 


*Papers  on  this  subject  were  given  by  the  writer  at  the  meeting  of  the 
American  Association  for  the  Advancement  of  Science  in  1879,  and  at  the 
meeting  of  the  Society  of  Naturalists  of  the  eastern  United  States  in  1883;  and 
in  Science  Vol.  Ill,  pp.  443,  444. 


204  PHOTO-MICROGRAPHY  [CH.    VIII 

|  272.  To  meet  all  the  difficulties  the  object  may  be  left  in  a  horizontal 
position  and  the  camera  made  vertical  (Fig.  161). 

Since  1879  such  a  camera  has  been  in  use  in  the  Anatomical  Department 
of  Cornell  University  for  photographing  all  kinds  of  specimens;  among  these, 
fresh  brains  and  hardened  brains  have  been  photographed  without  the  slightest 
injury  to  them.  Furthermore,  as  many  specimens  are  so  delicate  that  they 
will  not  support  their  own  weight,  they  may  be  photographed  under  alcohol 
or  water  with  a  vertical  camera  and  the  result  will  be  satisfactory  as  a  photo- 
graph and  harmless  to  the  specimen. 

A  great  field  is  also  open  for  obtaining  life-like  portraits  of  water  animals. 
Freshly  killed  or  etherized  animals  are  put  into  a  vessel  of  water  with  a  con- 
trasting back-ground  and  arranged  as  desired,  then  photographed.  Fins  have 
something  of  their  natural  appearance  and  gills  of  branchiate  salamanders 
float  out  in  the  water  in  a  natural  way.  In  case  the  fish  tends  to  float  in  the 
water  a  little  mercury  injected  into  the  abdomen  or  intestine  will  serve  as 
ballast. 

The  photographs  obtainable  in  water  are  almost  if  not  quite  as  sharp  as 
those  made  in  air.  Even  the  corrugations  on  the  scales  of  such  fishes  as  the 
sucker  (Catostomus  teres)  show  with  great  clearness.  Indeed  so  good  are  the 
results  that  excellent  half  tone  plates  may  be  produced  from  the  pictuers  thus 
made,  also  excellent  photogravures.  In  those  cases,  as  in  anatomical  prepara- 
tions, where  a  photograph  rarely  answers  the  requirements  of  a  scientific  figure, 
still  it  serves  as  a  most  admirable  basis  for  such  a  figure.  The  photograph  is 
made  of  the  desired  size  and  all  the  parts  are  in  correct  proportion  and  in  the 
correct  relative  position.  From  this  photographic  picture  may  be  traced  all 
the  outlines  upon  the  drawing  paper,  and  the  artist  can  devote  his  whole  time 
and  energy  to  giving  the  proper  expression  without  the  tedious  labor  of  mak- 
ing measurements. 

"While  the  use  of  photography  for  outlines  as  bases  for  figures  diminishes 
the  labor  of  artists  about  one-half  it  increases  that  of  the  preparator;  and  herein 
lies  one  of  its  chief  merits.  The  photographs  being  exact  images  of  the  prep- 
arations, the  tendency  will  be  to  make  them  with  greater  care  and  delicacy, 
and  the  result  will  be  less  imagination  and  more  reality  in  published  scientific 
figures;  and  the  objects  prepared  with  such  care  will  be  preserved  for  future 
reference." 

"  In  the  use  of  photography  for  figures  several  considerations  arise:  (i) 
The  avoidance  of  distortion;  (2)  The  adjustment  of  the  camera  to  obtain  an 
image  of  the  desired  size;  (3)  Focusing;  (4)  Lighting  and  centering  the  object. 

(i).  While  the  camera  delineates  rapidly,  the  image  is  liable  to  distortion. 
I  believe  opticians  are  agreed,  that,  in  order  to  obtain  correct  photographic 
images,  the  objective  must  be  properly  made,  and  the  plane  of  the  object 
must  be  parallel  to  the  plane  of  the  ground  glass.  Furthermore,  as  most  of 
the  objects  in  natural  history  have  not  plane  surfaces,  but  are  situated  in 
several  planes  at  different  levels,  the  whole  object  may  be  made  distinct  by 
using  in  the  objective  a  diaphragm  with  a  small  opening. 

$273.  Scale  of  Sizes  and  Focusing. — (2).  By  placing  the  camera  on  a 
long  table  and  a  scale  of  some  kind  against  the  wall,  the  exact  position  of  the 


CH.    VIII] 


-M1CROGRAPHY 


205 


Fig.  161.  Vertical  Camera  for  photographing  objects  in  a  horizontal  posi- 
tion. The  camera  is  attached  to  a  double  frame  connected  by  bent  metal  pieces 
fastened  to  the  lower  frame  and  sliding  in  a  groove  in  the  upper.  The  two 
frames  can  then  slide  over  each  other  without  separating.  For  moving  the 
outer  frame  a  rack  work  is  put  on  the  lower  or  inner  frame  and  a  pinion  with 
a  toothed  wheel  on  the  outer  one.  This  is  turned  by  the  wheel  shown.  To 
prevent  the  camera  running  down  in  the  vertical  position  a  pawl  is  held  in  place 
by  a  spring.  This  may  be  released  by  a  smaller  wheel  than  that  serving  to 
move  the  pinion.  This  rack  and  pinion  are  fine  enough  for  focusing  with  the 
photogaphic  objectives  employed. 

The  camera  bed  is  graduated  in  centimeters  so  that  the  exact  extent  of  the 
bellows  can  be  determined  by  inspection. 

The  support  on  ivhich  the  specimen  rests  is  of  heavy  glass  on  vertical  rods 
about  w  centimeters  long.  The  background  is  placed  on  the  table  top  about  10 
cm.  below.  This  arrangement  of  support  and  background  serves  to  avoid  the 
dense  shadows  which  make  it  difficult  to  determine  exactly  the  limits  of  the 
specimen.  To  make  the  apparatus  steady  the  right  hand  end  of  the  camera  table 
is  heavily  weighted.  The  tables  have  leveling  screws  in  the  legs. 


206 


PHO  TO-MICROGRA  PH ) ' 


\_CH.    VIII 


ground  glass  for  various  sizes  may  be  determined  once  for  all,  and  these  posi- 
tions noted  in  some  way. 

In  the  camera  here  figured,  the  camera  bed  is  ruled  in  centimeters  so  that 
the  position  of  the  ground  glass  can  be  determined  with  accuracy  and  noted. 
It  takes  but  a  moment  to  set  the  ground  glass  or  focusing  screen  at  the  right 
level  to  give  any  desired  size.  In  practice  it  is  convenient  to  have  attached  to 
the  camera  a  table  giving  the  position  of  the  ground  glass  for  various  sizes, 


Fig.  162  Beck's  Isosligmar  Objective.  "  The  lenses  are  ground  u<ith 
shallow  curvature  and  mounted  with  air  spaces  instead  of  cement  between  the 
individual  elements"'1  (Cut  loaned  by  Williams,  Brown  &  Earle,  Phila). 

and  also  the  distance  of  the  objective  from  the  object  in  each  case,  By  having 
this  information  it  takes  but  a  moment  to  set  the  camera  and  to  place  it  so 
that  it  will  be  approximately  in  focus.  The  final  focusing  is  then  accomplished 
by  the  use  of  the  rack  and  pinion  movement.  It  is  an  advantage  to  use  a 
focusing  glass  and  a  clear  focusing  screen  or  the  transparent  part  of  the  ordi- 
nary screen  (Fig.  163),  for  the  final  focusing.  Since  many  objects  have  no 
sharp  details  which  one  can  focus  on,  it  is  helpful  to  focus  on  some  printed 
letters  put  on  the  part  to  be  brought  out  with  the  greatest  sharpness.  Of 
course  these  are  removed  before  the  exposure  is  made. 


Fig.  163.  Ground-glass  focusing 
screen  u'ith  central  transparent  area  for 
exact  focusing  ivith  a  focusing  glass  when 
one  does  not  possess  a  clear  focusing  screen. 
(/)  The  groimd  surface;  (2}  Central 
part  with  oblong  cover-glass  over  Canada 
balsam  on  the  ground  surface  to  render  it 
transparent.  X.  The  central  point  in 
the  entire  focusing  screen;  It  ^s  made 
with  a  black  lead  pencil  on  the  ground 
surface.  The  focusing  glass  is  focused  on 
this  cross,  then  when  the  image  is  in  focus 
it  will  be  at  the  level  of  the  sensitive 
coating  of  the  plate. 


\  274.     In  Lighting  the  object  one  should  take  pains  to  so  arrange  it  with 
reference  to  the  light  that  the  details  will  show  with  the  greatest  clearness. 


CH.    /'///]  PHOTO-MICROGRAPHY  207 

Naturally  for  the  vertical  camera  the  light  will  come  from  the  side  and  not 
from  a  skylight,  although  good  results  are  obtained  with  a  skylight  if  one  so 
places  the  camera  that  it  does  not  cast  objectionable  shadows. 

As  shown  in  Figs.  161,  165,  the  object  is  placed  upon  a  glass  support  and 
the  background  is  quite  a  distance  below  the  support.  For  a  dark  object  the 
background  should  be  light,  and  for  a  light  one  dark.  Black  velveteen  is 
excellent  for  a  back-ground.  The  advantage  of  the  glass  support  is  that  the 
shadows  in  the  background  which  often  make  it  difficult  to  tell  just  where  the 
specimen  ends  and  the  background  begins,  are  wholly  done  away  with,  and 
that  too  without  at  all  affecting  the  proper  light  and  shade  of  the  object  itself. 
(Method  of  W,  E.  Rumsey,  Canadian  Entomologist  1896,  p.  84). 


Fig  164.      Tripod  magnifier  as  a  focusing  glass. 

This  is  carefully  focused  on  a  scratch  or  pencil  mark 
on  (lie  lower  or  ground  surface  of  the  focusing  screen. 

Then  whenever  the  object  is  sharply  focused  the  focal 
plane  u'ill  be  at  the  level  of  the  sensitive  surface. 


\  275.  Prints. — If  the  photographic  prints  are  to  be  used  solely  for  out- 
lines, the  well-known  blue  prints  so  much  used  in  engineering  and  architecture 
may  be  made.  If,  however,  light  and  shade  and  fine  details  are  to  be  brought 
out  with  great  distinctness,  either  an  aristotype,  velox,  platinotype  or  bromide 
print  is  preferable. 

§  276.  Recording,  Storing  and  Labeling  Negatives. — In 
order  to  get  the  greatest  benefit  from  past  experience  it  is  necessary 
to  make  the  results  available  by  means  of  a  careful  record.  For 
this  purpose  the  table  (§  316)  has  been  prepared.  If  one  gives  the 
information  called  for  in  this  table,  whether  the  result  is  successful 
or  not,  one  can  after  a  time  work  with  great  exactness,  for  the 
elements  of  success  and  failure  will  stand  out  clearly  in  the  table. 

§  277.  Labeling  Negatives. — After  a  negative  is  dry  the 
labeling  can  be  done  on  the  gelatin  side  with  carbon  ink. 
Enough  data  should  be  given  to  enable  the  certain  identification  of 
the  negative  at  any  future  time. 

§  278.  Storing  Negatives. — This  is  satisfactorily  done  by 
putting  each  into  an  envelope  and  writing  a  duplicate  label  on  the 
upper  edge,  and  then  the  negatives  may  be  placed  in  drawers  in 


208 


PHO  TO-MICROGRAPHY 


[CH.    VIII 


t  FIG.  165.  Vertical  Camera  and  special  microscope  stand  for  photograph- 
ing embryos  and  other  small  specimens  in  liquids  and  for  photographing 
large  sections.  The  camera  rests  on  a  low  table  and  the  operator  can  stand  on 
the  floor  while  performing  all  the  manipulations. 

The  stage  of  the  microscope  is  attached  to  the  arm  in  the  place  of  the  tube. 
This  stage  has  tzvo  stories.  The  specimen  is  shown  on  the  upper  and  the  back- 
ground on  the  lower  story.  In  focusing,  the  coarse  and  fine  adjustment  of  the 
special  microscope  stand  are  used.  The  extention  of  the  camera  for  a  definite 
size  of  picture  is  discussed  in  \  273. 


CH.    J7/7]  rilOTO-MICKOGRAPHY 


209 


alpliabetical  order  as  are  the  catalog  cards  of  books  in  a  library. 
One  can  then  find  any  negative  with  the  same  facility  that  the  title 
of  a  book  can  be  found  in  a  card  catalog. 

PHOTOGRAPHING    KMHRYOS 

For  photographing  embryos  and  many  other  small  specimens 
it  is  more  convenient  to  use  a  smaller  apparatus  than  the  vertical 
camera  just  described.  It  is  necessary  also  to  have  a  more  delicate 
method  of  focusing. 

§  279.  Camera  for  Embryos.— This  is  a  vertical  camera  for 
photographing  with  the  microscope,  and  with  a  photographic  objec- 
tive in  the  end  of  the  camera  as  for  an  ordinary  camera.  This  is 
readily  accomplished  by  having  a  society  screw  adapter,  and  also 
adapters  for  the  micro-planars  or  other  objectives  which  one  desires 
to  use.  The  magnification  usually  required  varies  from  natural  size 
(X  i)  to  five  times  natural  size  (X  5)  up  to  X  20.  As  with  the 
large  camera  the  position  of  the  ground  glass  for  each  magnification 
and  for  each  objective  is  determined  once  for  all  by  using  a  scale  in 
millimeters.  The  various  positions  are  accurately  noted,  then  one 
can  set  the  camera  almost  instantly  for  the  desired  magnification. 
The  supporting  rod  is  divided  to  half  centimeters  and  therefore  the 
exact  position  is  easily  recorded  (Fig.  172). 


FIG.  166.  Diagram  of  the  general 
construction  of  -the  Micro-Tcssar  ob- 
jectives of  the  Bausch  &  Lomb  Optical 
Co. 


§  280.  Special  Microscope  Stand. — For  the  accurate  focus- 
ing necessary  for  embryos  one  should  possess  a  special  microscope 
stand  with  the  stage  in  two  stories  and  attached  to  the  arm  in  place 
of  the  tube  of  the  microscope.  The  stage  proper  is  absent.  This 
arrangement  of  the  stage  permits  the  use  of  the  coarse  and  fine 
adjustment  of  the  microscope  to  be  used  for  focusing.  The  position 
of  the  camera  on  a  low  table  (45  to  50  cm.  high)  makes  it  possible 
for  the  operator  to  stand  on  the  floor  while  making  all  the  adjust- 
ments of  the  embryo  and  for  focusing;  and  all  the  parts  are  within 
reach  (Fig.  165). 


PHO  TO-MICROGRAPI1 ) 


\_CH.    VIII 


§281.  Arranging  the  Embryos.  — As  usually  prepared  the 
embryos  are  white  and  therefore  require  a  dark  background.  This 
may  be  attained  either  by  placing  the  embryos  in  a  dark  dish  or  on 
some  paper  blackened  with  water-proof  India  ink,  or  by  putting  them 
in  a  glass  vessel  like  a  Petri  dish  with  a  piece  of  black  velveteen  on 
the  stage  below.  Black  glass  on  the  bottom  of  the  dish  containing 
the  embryos  etc.,  forms  one  of  the  best  dark  backgrounds.  The 
specimens  will  of  course  be  in  a  liquid,  usually  alcohol. 


FIG.  167-178.  Lcitz  Micro- 
summar  Objectives  0/64  and  42 
mm.  focus.  (Cuts  from  Leitz 
Photomicrographic  catalog}. 


If  several  embryos  are  to  be  taken  at  once,  they  are  arranged 
in  rows  something  as  the  words  on  a  line.  Arrange  them  in 
even  vertical  as  well  as  horizontal  rows  so  that  when  the  print  is 
made  it  will  be  easy  to  cut  them  apart.  When  the  embryos  are 
arranged,  one  should  be  certain  that  the  light  brings  out  the  details 
most  desired.  For  example,  if  one  is  photographing  an  embryo 
which  shows  the  branchial  pockets  well,  great  pains  should  be  taken 
to  so  arrange  the  embryo  with  reference  to  the  light  that  the  proper 
shading  will  be  given  to  bring  out  the  gill  pockets  most  emphati- 
cally. One  can  learn  to  do  this  only  by  practice.  It  is  advanta- 
geous to  have  an  assistant,  then  while  the  operator  is  looking  into 
the  camera  the  assistant  can  turn  the  embryo  in  various  directions 
until  the  appearance  is  most  satisfactory. 

§  282.  Focusing. — For  getting  a  general  focus,  and  for  the 
general  arrangement  the  ground  glass  screen  is  used,  but  for  the 
final  focusing  it  is  desirable  to  use  a  focusing  glass. 

The  tripod  microscope  answers  fairly  well  for  a  focusing  glass, 
but  several  are  now  made  with  much  more  perfect  corrections  and 
for  photo-micrography  it  is  desirable  to  have  as  good  apparatus  as 
can  be  obtained.  For  using  the  focusing  glass  one  may  have  a  clear 
glass  screen  and  set  the  focusing  glass  upon  it.  There  should  be  a 
diamond  cross  in  the  middle  of  the  screen  on  the  under  side  where 
the  ground  surface  usually  is  and  this  surface  of  the  glass  like  that 


CII.    /7//]  PHOTO-1\[ICR(H;RAPIIY 


of  the  ground  surface  must  be  exactly  at  the  level  where  the  sensi- 
tive film  of  the  plate  is  in  taking  the  picture.  Focus  the  focusing 
glass  accurately  upon  the  diamond  scratch  and  fix  the  glass  so  that 
it  will  remain  at  exactly  that  distance  from  the  diamond  scratch. 
Then  when  an  object  is  to  be  focused  if  the  image  is  perfectly  sharp 
under  the  focusing  glass  its  real  image  will  be  at  the  proper  level 
for  taking  the  picture. 

A  still  more  satisfactory  method  for  the  final  focusing  is  to  have 
one  of  the  better  forms  of  focusing  glass  mounted  in  a  board  screen, 
then  one  looks  at  the  aerial  image  formed  by  the  objective  exactly 
as  in  looking  into  a  microscope.  One  must  take  especial  pains  in 
setting  the  focusing  glass  in  the  board  screen  so  that  the  real  image 
will  be  at  the  right  level.  One  can  do  this  by  placing  a  ground 
glass  in  the  plate  holder  and  putting  it  in  position  on  the  camera 
and  then  focusing  some  printed  letters  as  sharply  as  possible.  This 
will  get  the  real  image  at  exactly  the  right  level  for  the  plate  holder 
to  be  used  in  making  the  picture.  Then  the  board  screen  with  the 
focusing  glass  is  put  in  place  of  the  plate  holder  and  the  focusing 
glass  moved  up  and  down  until  the  image  is  as  sharp  as  possible. 
The  focusing  glass  is  then  fixed  in  position,  and  any  object  focused 
with  it  should  be  equally  in  focus  on  the  sensitive  film  for  making 
the  picture. 

This  method  has  the  great  advantage  that  there  is  nothing 
between  the  focusing  glass  and  the  aerial  image,  and  one  can  focus 
as  easily  and  certainly  in  this  way  as  with  a  compound  microscope. 
For  this  final  focusing  it  is  better  to  have  the  diaphragm  opening 
as  it  is  to  be  in  taking  the  picture,  although  for  getting  the  general 
focus  and  arranging  the  object  when  the  ground  glass  is  in  position 
the  full  opening  of  the  objective  may  be  used  for  the  greater 
illumination. 

§  283.  Exposure.  —  In  daylight  with  white  embryos  and  a 
dark  ground  30  to  40  seconds  is  usually  sufficient  exposure.  One 
must  learn  this  also  by  trial  and  it  facilitates  the  obtaining  of  exact 
data  to  make  a  record  of  every  negative  made,  whether  the  negative 
is  good  or  bad.  A  table  is  given  in  §  316  to  facilitate  the  record 
taking.  In  a  short  time  one  can  learn  to  make  the  correct  exposure. 
If  the  result  is  unsatisfactory,  try  again.  The  rule  adhered  to  by 
all  first  rate  workers  is  to  stick  to  it  until  the  result  is  satisfactory. 

§  284.     Records  of  Embryos.  —  Each  specimen  or  group  of 


PHO  TO-MICROGRAPHY 


[CY/.    VIII 


specimens  will  have  its  own  label  giving  date  and  method  of  prepar- 
ation. It  is  an  advantage  to  write  this  label  with  water- proof  car- 
bon ink,  then  one  can  put  the  label  in  the  dish  with  the  embryos 
and  it  will  form  a  part  of  the  picture  and  serve  as  a  record. 

After  the  picture  is  satisfactorily  made  it  is  wise  to  number  the 
embryos  on  the  back  of  the  negative  with  a  wax  crayon,  and  later 
when  the  negative  is  dry  number  on  the  front  with  carbon  ink. 
The  embryos  are  placed  in  separate  bottles  each  with  a  copy  of  the 
original  label  and  the  number  corresponding  with  that  put  on  the 
negative.  This  is  easily  accomplished  if  the  embryos  are  arranged 
in  definite  rows  as  advised  in  §  281. 


FIG.  169.  Camera  and  special  microscope  stand  for  photographing  large 
transparent  sections.  For  this  the  vertical  camera  is  used  (Fig.  161}  icith  the 
camera  reversed  on  the  sliding  frame.  This  frame  is  elevated  sufficiently  to 
utilize  the  sky  as  background  and  illuminant.  The  special  microscope  stand 
is  inclined  to  the  horizontal  and  placed  on  the  fixed  frame  supporting  the 
camera;  the  specimen  placed  on  the  stage.  For  objective  one  of  those  objectives 
shoitn  in  Figs.  162,  166-168  is  used.  The  objective  is  screwed  into  an  adapter 
in  place  of  the  ordinary  photographic  objective.  The  focusing  is  performed 
roughly  by  the  rack  and  pinion,  and  then  with  great  exactness  with  the  focus- 
ing glass.  For  manipulating  the  fine  adjustment  of  the  special  microscope  the 
well  known  device  of  a  cord  over  the  head  of  the  micrometer  screw  is  used. 
(See  also  Fig.  /6j.)  (  Trans.  Amer.  Micr.  Soc., 


ClI.    VIII]  rilOTO-MlCROGRAPHY  213 

Finally  when  the  embryo  is  cut  into  serial  sections  and 
mounted,  a  picture  of  the  whole  embryo  should  accompany  the 
series. 

£  285.  Size  of  the  Pictures. — For  all  embryos  it  is  well  to 
make  one  picture  natural  size  (  X  i )  and  then  for  the  smallest  ones 
a  magnification  of  at  least  five  times  natural  size  (X  5).  Here,  as 
with  the  magnification  of  the  microscope,  linear  magnification  is 
always  meant  (§  170,  171). 

§  286.  Objectives. — For  making  pictures  from  one  to  five 
times  natural  size  objectives  of  60  to  100  mm.  focus  answer  well 
(Figs.  166-167).  Short  focus  (75  to  100  mm.  equivalent  focus), 
wide  angle  photographic  objectives  are  also  admirable  for  this  work. 

>;  287.  Record  of  Negatives. — As  indicated  in  §  276-278  each 
negative  should  have  a  record,  see  record  blank  (§  316).  On  the 
negative  itself  should  be  also  written  the  main  facts  with  carbon  ink. 
The  name  and  magnification,  date  and  any  other  details  which  may 
be  thought  desirable  can  be  put  on  the  envelope  containing  the  nega- 
tive and  then  stored  like  a  catalog  card  as  described  above  (§  278). 

S  288.  Photographing  Large  Transparent  Objects.— 
There  are  many  large  transparent  objects  which  it  is  desirable  to 
photograph,  e.  g.,  chick  embryos  mounted  whole,  large  sections  of 
organs  like  the  brain,  etc.  These  must  be  photographed  at  a  low 
magnification. 

Successful  photographs  require  an  even  lighting  and  an  objec- 
tive which  has  sufficient  field  to  take  in  the  whole  object.  The 
camera  used  for  embryos  (Fig.  182)  may  be  used  in  connection  with 
the  projection  microscope  condenser.  For  very  large  objects  or  for 
large  pictures  the  vertical  camera  (Figs.  161,  169)  is  reversed  in 
position  on  the  supporting  frames,  and  elevated  only  sufficiently  to 
make  a  sky  back-ground  ;  or  a  45  degree  reflector  of  white  cloth  or 
paper  of  sufficient  size  must  be  used  fora  horizontal  camera.  If  one 
has  the  earth  for  back-ground  the  light  will  be  dull  and  uneven 
and  a  very  long  exposure  is  necessary,  and  the  final  results 
unsatisfactory. 

§  289.  Use  of  the  Special  Microscope  Stand. — In  order  to 
hold  the  specimen  in  position  and  to  focus  it  accurately,  it  is  put  on 
the  stage  of  the  special  microscope  stand  (Fig.  165),  which  is 


214  PHOTO-MICROGRAPHY  [CH.   VIII 

inclined,  and  fastened  to  the  fixed  part  of  the  frame  supporting  the 
camera.  As  the  stage  of  this  microscope  is  moved  by  the  coarse  or 
the  fine  adjustment,  the  focusing  can  be  accomplished  with  the  same 
accuracy  as  the  microscope  itself.  For  the  general  arrangement  of 
the  specimen  and  the  rough  focusing  the  ground  glass  is  used,  then 
this  is  replaced  by  a  clear-glass  focusing  screen,  and  by  the  aid  of  a 
focusing  glass  the  specimen  is  put  in  perfect  focus.  (See  also  §  282.) 
As  one  cannot  reach  the  fine  adjustment  while  focusing,  the  well 
known  device  of  a  cord  over  the  head  of  the  micrometer  screw  is 
resorted  to.  The  two  ends  of  the  cord  should  be  weighted  with 
about  50  or  a  hundred  grams  to  keep  the  cord  taut,  then  whichever 
one  is  pulled,  the  micrometer  screw  will  respond  at  once.  To  cut 
off  the  light  a  piece  of  black  velveteen  is  hung  over  the  end  of  the 
objective.  This  can  be  removed  without  jarring  the  apparatus. 
An  exposure  of  a  few  seconds  (3  to  10  seconds),  will  suffice  for 
many  preparations,  unless  a  color  screen  is  used.  The  color  screen 
increases  the  time  of  exposure  (§  291). 

COLOR-CORRECT    PHOTOGRAPHY 

From  the  fact  that  the  different  wave  lengths  of  light  affect  the  photo- 
graphic plate  with  different  degrees  of  vigor,  the  ordinary  photographic  print 
of  many-colored  objects  or  landscapes  is  not  satisfactory.  All  objects  whose 
light  is  of  short  wave  lengths,  as  blue,  etc.,  will  appear  too  light  and  those 
with  greater  wave  lengths  as  red,  yellow  and  green  will  be  too  dark  relatively. 
To  obviate  this  difficulty  two  methods  have  been  adopted,  and  for  the  most 
complete  success  they  must  be  combined. 

(A)  The  use  of  ortho-  or  iso-chromatic  plates  and  (B)  the  use  of  a  color 
screen  or  light  filter. 

|  290.  Orthochromatic  or  Isochromatic  Plates. — These  ar£  plates  which 
have  been  rendered  much  more  sensitive  than  ordinary  plates  to  the  long 
waves  of  red,  orange,  yellow  and  green,  they  therefore  give  a  much  more 
natural  rendering  to  many-colored  objects  than  ordinary  plates  While  color- 
sensitizing  has  been  carried  to  a  considerable  stage  of  perfection  and  there  is 
a  large  choice  of  plates  now  on  the  market  for  special  purposes,  it  should  be 
remembered  that  no  matter  for  what  color  or  colors  a  plate  has  been  sensitized 
it  remains  more  sensitive  to  the  short  waves  (violet  end  of  spectrum]  than  to 
the  long  waves  (red  end  of  spectrum}.  Therefore  to  obtain  a  correct  rendering 
of  variously  colored  objects  by  photography,  a  color  screen  is  necessary  as 
well  as  a  color  sensitive  plate  (  \  291) . 

These  color-correct  plates  are  not  very  enduring,  and  must  be  used  while 
they  are  fresh,  or  only  weak,  foggy  negatives  will  result;  and  as  they  are  sensi- 
tive to  orange,  etc.,  one  must  be  very  careful  in  exposing  them  in  the  dark 


CH.    17/1]  PHOTO-MICROCRAPHY  215 

room  even  to  the  light  of  the  developing  lantern.  The  more  nearly  the  plate 
can  be  kept  from  all  the  light,  except  that  acting  during  the  exposure  in  the 
camera,  the  more  satisfactory  will  be  the  resulting  negative. 

2  291.  Requirements  for  Successful  Photo-Micrography. — Successful  vis- 
ual images  may  be  obtained  in  two  ways,  ( \  $  118-119,  I57-J59).  viz:  by  mount- 
ing the  object  in  a  medium  whose  refractive  index  differs  markedly  from  the 
object;  or  by  staining  the  object  so  that  it  has  a  markedly  different  color  from 
the  mounting  medium.  When  the  two  methods  are  combined  and  the  object 
differs  both  in  refractive  index  and  in  color  from  the  mounting  medium  the 
visual  images  obtained  through  the  microscope  are  most  satisfactory. 

In  photography  the  difference  in  refractive  index  between  object  and 
surrounding  medium  is  of  the  same  importance  as  for  ordinary  observation, 
and  as  with  the  eye,  the  greater  the  difference  the  bolder  the  outline  (?  157). 
But  difference  in  color  of  object  and  mounting  medium  does  not  ensure  a  good 
photographic  image.  This  is  because  the  wave  lengths  of  light  producing 
the  different  colors  are  not  all  equally  effective  in  producing  a  photograph. 
The  visually  brilliant  long  waves  of  red,  orange,  yellow  and  green  are  far  less 
effective  in  producing  a  picture  than  the  shorter  waves  of  blue,  indigo  and 
violet.  In  a  word  the  end  of  the  spectrum  brightest  to  the  eye  is  least  effec- 
tive for  producing  a  photograph,  i.e.  the  sensitiveness  of  the  eye  and  the  pho- 
tographic plate  are  inverted  or  complementary. 

As  stated  above  (g  290)  color  sensitized  plates  have  been  produced  to  meet 
a  part  of  the  difficulty.  To  further  perfect  the  photographic  image  and  make 
it  correspond  more  closely  with  the  light  effects  of  the  visual  image,  color- 
screens  or  filters  have  been  devised  whereby  the  light  transmitted  by  the 
specimen  is  partly  or  wholly  eliminated  from  the  light  illuminating  it.  If  the 
color  screen  wholly  eliminates  the  light  which  the  object  transmits  then  of 
course  its  color  to  the  eye  is  eliminated  and  the  object  appears  black,  no  mat- 
ter how  brilliant  the  illumination.  It  is  also  black  to  the  photographic  plate, 
and  shows  as  a  black  object  in  the  picture. 

The  dyes  used  in  staining  microscopic  preparations  differ  not  only  in  the 
wave  length  of  light  they  allow  to  pass  through  the  object,  but  they  also  differ 
in  the  amount  of  opacity  to  all  light  which  they  give  to  the  specimen.  This 
is  a  valuable  feature  for  photography.  For  example  hematoxylin  transmits 
much  actinic  light,  but  it  also  renders  the  object  more  or  less  opaque  to  all 
light  and  hence  specimens  well  stained  in  hematoxylin  usually  give  good 
photographs.  Carmine  stained  specimens  also  give  good  photographs  because 
they  are  rendered  slightly  opaque  to  all  light,  but  principally  because  the  red 
stain  is  especially  opaque  to  the  short  waves.  If  one  could  select  stains  for  all 
objects  which  would  greatly  lessen  the  passage  of  all  light,  the  amount  cutout 
depending  on  the  density  of  the  specimen  in  different  parts,  and  also  elimi- 
nate the  greater  number  of  the  short  waves,  it  would  be  easy  to  produce  good 
photo-micrographs.  Where  stains  with  the  above  qualities  cannot  be  employed 
it  is  necessary  not  only  to  use  isochromatic  plates  but  a  proper  color-screen. 

$  292.  Color  Screens. — For  the  intelligent  use  of  color-screens  it  must  be 
borne  in  mind  that  colored  objects  appear  colored  to  the  eye  because  they 


2 16  PHOTO-MICROGRAPHY  [CH.    VIII 

transmit  certain  wave  lengths  of  light  and  absorb  others.  If  the  color  is  a 
pure  orange  for.  example  all  the  other  colors  of  the  spectrum  have  been  absorbed 
by  the  object  (\  \  214-217).  Usually,  however  a  greater  or  less  number  of  waves 
of  other  colors  are  transmitted  also. 

If  one  wishes  to  get  the  greatest  possible  contrast  in  photographing  an 
object  stained  with  pure  orange  a  color  screen  is  used  transmitting  all  the  other 
colors  except  orange.  Then  as  the  object  can  transmit  only  orange  light  it 
absorbs  all  the  light  sent  to  it  while  on  all  sides  of  the  object  light  of  all  the 
other  wave  lengths  will  reach  the  photographic  plate  and  affect  it,  hence  in 
the  photograph  the  orange  object  appears  black  in  a  light  field. 

Although  objects  seen  in  the  microscope  may  appear  of  a  certain  color 
they  usually  transmit  also  wave  lengths  of  other  colors  so  that  there  is  a  cer- 
tain amount  of  detail  shown  in  the  picture  due  to  the  different  amounts  of 
effective  light  waves  which  are  transmitted  in  different  parts  depending  upon 
the  varying  density  of  the  object. 

Where  there  is  no  detail  as  with  many  bacteria,  the  blacker  the  object 
appears  in  the  picture  the  better,  hence  in  such  cases  a  monochromatic  color 
screen  complementary  to  the  color  transmitted  by  the  bacteria  or  other  objects 
would  give  the  most  satisfactory  results. 

Proper  choice  of  a  color  filter  is  greatly  aided  by  studying  the  object  to 
be  photographed  with  a  micro-spectroscope  to  see  what  wave  lengths  and  the 
proportion  of  each  are  actually  transmitted  by  the  specimen.  Then  if  one 
studies  the  color-screens  available  he  will  be  able  to  select  the  one  most  nearly 
complementary  to  the  object  to  be  photographed.  As  stated  above,  it  is  desir- 
able in  histologic  preparations  with  structural  detail  to  show  such  detail. 
This  is  partly  determined  by  the  different  refractive  index  of  the  different 
parts,  and  it  can  be  greatly  accentuated  by  selecting  a  color  screen  which 
eliminates  the  excess  of  the  short  waves  from  the  light.  For  many  objects 
stained  with  dyes  giving  strong  contrast  etc.  as  hematoxylin  and  carmine, 
good  pictures  may  be  obtained  without  a  color  screen  if  isocbromatic  plates 
are  employed  and  a  kerosene  lamp  is  used  for  illumination.  The  kerosene 
light  is  very  rich  in  the  waves  near  the  middle  of  the  spectrum,  but  rather 
poor  in  the  short  waves. 

£  293.  Composition  and  Preparation  of  Color  Screens. — In  recent  years 
as  the  principles  for  the  proper  selection  and  use  of  color  screens  have  become 
more  fully  understood  the  range  has  been  greatly  increased  of  the  appropriate 
color-sensitive  plates.  While  color  screens  of  solutions  are  still  used,  perhaps 
the  majority  of  screens  now  employed  are  made  of  variously  colored  glass  or 
of  glass  coated  with  variously  stained  gelatin  or  collodion. 

By  recalling  the  work  with  the  spectroscope  (2  217),  it  will  be  remem- 
bered that  the  light  transmitted  through  a  colored  object  depends  upon  the 
thickness  of  the  object  and  also  upon  the  intensity  of  the  illumination.  This 
being  true  the  same  color  screen  may  be  made  to  give  greater  or  less  contrast 
in  the  photograph  by  varying  the  intensity  of  the  illumination.  If  one  studies 
the  spectrum  of  solutions  of  the  Various  dyes  used  in  microscopy,  like 
aurantia,  methyl  green,  etc.,  he  can  select  colors  for  his  color  screen  which 
give  contrast  for  the  specimens  he  has  to  photograph,  remembering  always 


CH.   VIII]  PHOTO-VICRO(;RAPHY  217 

that  the  screen  should  cut  out  much  of  the  blue  end  of  the  spectrum  and  also 
the  special  color  transmitted  by  the  object. 

Alcoholic  solutions  of  the  dye  chosen  may  then  be  used  to  stain  collodion 
(see  Ch.  IX)  or  either  alcoholic  or  aqueous  solutions  may  be  used  for  staining 
glass  plates  coated  with  20%  to  30  ';<  gelatin. 

1  294.  Position  and  Exposure  with  Color-Screens. — It  does  not  make 
much  difference  where  the  color  screen  is  placed  provided  no  light  reaches 
the  plate  which  has  not  passed  through  it.  The  most  convenient  position  is 
between  the  source  of  light  and  the  object.  If  one  uses  a  glass  screen  or 
screens  of  stained  collodion  or  gelatin  on  glass,  the  most  convenient  position 
is  in  the  holder  for  the  central  stop  diaphragms  just  under  the  condenser. 

The  length  of  exposure  required  when  color  screens  are  used  is  ordinarily 
considerably  increased.  For  color  sensitive  plates  the  increase  is  greatly 
lessened  if  screen  and  plate  are  mutually  adapted. 

PHOTOGRAPHING    WITH    A    MICROSCOPE* 

\  295.  The  first  pictures  made  on  white  paper  and  white  leather,  sensiti/.ed 
by  silver  nitrate,  were  made  by  the  aid  of  a  solar  microscope  (1802).  The 
pictures  were  made  by  Wedgewood  and  Davy,  and  Davy  says  :  "I  have  found 
that  images  of  small  objects  produced  by  means  of  the  solar  microscope  may 
be  copied  without  difficulty  on  prepared  paper,  "f 

"Considerable  confusion  exists  as  to  the  proper  nomenclature  of  photography 
with  the  microscope.  In  German  and  French  the  term  micro-photography  is 
very  common,  while  in  English  photo-micrography  and  micro- photography 
mean  different  things.  Thus:  A  photo-micrograph  is  a  photograph  of  a  small 
or  microscopic  object  usually  made  \\ith  a  microscope  and  of  sufficient  size 
for  observation  with  the  unaided  eye;  while  a  micro-photograph  is  a  small  or 
microscopic  photograph  of  an  object,  usually  a  large  object,  like  a  man  or 
woman  and  is  designed  to  be  looked  at  with  a  microscope. 

Dr.  A.  C.  Mercer,  in  an  article  in  the  Proc.  Amer.  Micr.  Soc. ,  1886,  p.  131, 
says  that  Mr.  George  Shadbolt  made  this  distinction.  See  the  Liverpool  and 
Manchester  Photographic  Journal  (now  British  Journal  of  Photography},  Aug. 
15,  1858,  p.  203;  also  Button's  Photographic  Notes,  Vol.  Ill,  1858,  pp.  205-208. 
On  p.  208  of  the  last,  Shadbolt's  word  "Photomicrography"  appears.  Dr. 
Mercer  puts  the  case  very  neatly  as  follows:  "A  Photo- J\ficrograph  is  a 
macroscopic  photograph  of  a  microscopic  object;  a  micro-photograph  is  a 
microscopic  photograph  of  a  macroscopic  object.  See  also  Medical  News,  Jan. 
27,  1X94,  p.  108. 

tin  a  most  interesting  paper  by  A.  C.  Mercer  on  "The  Indebtedness  of 
Photography  to  Microscopy,"  Photographic  Times  Almanac,  1887,  it  is  shown 
that  :  "  To  briefly  recapitulate,  photography  is  apparently  somewhat  indebted 
to  microscopy  for  the  first  fleeting  pictures  of  Wedgewood  and  Davy  [1802], 
the  first  methods  of  producing  permanent  paper  prints  [Reede,  1837-1839], 
the  first  offering  of  prints  for  sale,  the  first  plates  engraved  after  photographs 


2i8  PHOTO-MICROGRAPHY  [CH.   VIII 

Thus  among  the  very  first  of  the  experiments  in  photography  the  micro- 
scope was  called  into  requisition.  And  naturally  plants  and  motionless  objects 
were  photographed  in  the  beginnings  of  the  art  when  the  time  of  exposure 
required  was  very  great. 

At  the  present  time  photography  is  used  to  an  almost  inconceivable  degree 
in  all  the  arts  and  sciences  and  in  pure  art.  Even  astronomy  finds  it  of  the 
greatest  assistance. 

It  has  also  accomplished  marvels  in  the  production  of  colored  plates  for 
book  illustrations,  especially  in  natural  history.  For  an  example  see  Corn- 
stock's  Insect  Life",  2d  edition. 

Although  first  in  the  field,  Photo-Micrography  has  been  least  successful  of 
the  branches  of  photography.  This  is  due  to  several  causes.  In  the  first 
place,  microscopic  objectives  have  been  naturally  constructed  to  give  the 
clearest  image  to  the  eye,  that  is  the  visual  image  as  it  is  sometimes  called,  is 
for  microscopic  observation,  of  prime  importance.  The  actinic  or  photo- 
graphic image,  on  the  other  hand,  is  of  prime  importance  for  photography. 
For  the  majority  of  microscopic  objects  transmitted  light  (\  73)  must  be  used, 
not  reflected  light  as  in  ordinary  vision.  Finally,  from  the  shortness  of  focus 
and  the  smallness  of  the  lenses,  the  proper  illumination  of  the  object  is 
accomplished  with  some  difficulty,  and  the  fact  of  the  lack  of  sharpness  over 
the  whole  field  with  any  but  the  lower  powers,  have  combined  to  make  photo- 
micrography less  successful  than  ordinary  macro-photography.  So  tireless, 
however,  have  been  the  efforts  of  those  who  believed  in  the  ultimate  success 
of  photo-micrography,  that  now  the  ordinary  achromatic  objectives  with 
ortho-chromatic  or  isochromatic  plates  and  a  color  screen  or  petroleum  light 
give  good  results,  while  the  apochromatic  objectives  with  projection  oculars 
give  excellent  results,  even  in  hands  not  especially  skilled.  The  problem  of 
illumination  has  also  been  solved  by  the  construction  of  achromatic  and 
apochromatic  condensers  and  by  the  electric  and  other  powerful  lights  now 
available.  There  still  remains  the  difficulty  of  transmitted  light  and  of  so  pre- 
paring the  object  that  structural  details  stand  out  with  sufficient  clearness  to 
make  a  picture  which  approaches  in  definiteness  the  drawing  of  a  skilled 
artist. 

The  writer  would  advise  all  who  wish  to  undertake  photo-micrography 
seriously,  to  study  samples  of  the  best  work  that  has  been  produced.  Among 
those  who  showed  the  possibilities  of  photo-micrographs  was  Col.  Woodward 
of  the  U.  S.  Army  Medical  Museum.  The  photo-micrographs  made  by  him 
and  exhibited  at  the  Centennial  Celebration  at  Philadelphia  in  1876,  serve 
still  as  models,  and  no  one  could  do  better  than  to  study  them  and  try  to  equal 
them  in  clearness  and  general  excellence.  According  to  the  writer's  observa- 
tion no  photo-micrographs  of  histologic  objects  have  ever  exceeded  those 


for  the  purpose  of  book  illustration  [Donne  &  Foucault,  1845],  the  photo- 
graphic use  of  collodion  [Archer  &  Diamond,  1851],  and  finally,  wholly 
indebted  for  the  origin  of  the  gelatino-bromide  process,  greatest  achievement 
of  them  all  [Dr.  R.  L,.  Maddox,  1871].  See  further  for  the  history  of  Photo- 
micrography, Neuhauss,  also  Bousfield. 


CH. 


PHOTO-MICROGRAPH  > 


219 


made  by  Woodward,  and  most  of  them  are  vastly  inferior.  It  is  gratifying  to 
state,  however,  that  at  the  present  time  many  original  papers  are  partly  or 
wholly  illustrated  by  photo-micrographs,  and  no  country  has  produced  works 
with  photo-rnicrographic  illustrations  superior  to  those  in  "  Wilson's  Atlas  of 
Fertilization  and  Karyokinesis  "  and  ".Starr's  Atlas  of  Nerve  Cells,''  issued  by 
the  Columbia  University  Press. — 

In  passing  the  writer  would  like  to  pay  a  tribute  to  Mr.  W.  H.  Walmsley 
who  has  labored  in  advancing  photo -micrography  for  the  last  twenty  years. 
His  convenient  apparatus  and  abundant  experience  have  been  placed  freely  at 
the  command  of  every  interested  worker,  and  many  a  beginner  has  been 
helped  over  difficulties  by  him.  His  last  contribution  in  "International 
Clinics,"  vol.  i.  ser.  n,  12,  is  encouraging  in  the  highest  degree  both  for  its 
matter  and  for  the  illustrations. 


FIG.  170.  Zeiss*  Vertical  Photo-micro- 
graphic  Camera.  A.  Set  screw  holding 
the  rod  (5)  in  any  desired  position.  P.  Q. 
Si' I  screws  by  which  the  belloivs  are  held  in 
place.  B .  Stand  with  tripod  base  in  which 
the  supporting  rod  (S)  is  held.  This  rod  is 
now  graduated  in  centimeters  and  is  a 
ready  means  of  determining  the  length  of 
the  camera.  M.  Mirror  of  the  microscope. 
L.  The  sleeve  serving  to  make  a  light- 
tight  connection  between  the  camera  and 
microscope.  O.  The  lower  end  of  the 
camera.  R.  '1  he  upper  end  of  the  camera 
where  the  focusing  screen  and  plate  holder 
are  situated.  (From  Zeiss'  Photo-micro- 
graphic  Catalog.} 


As  the  difficulties  of  photo-micrography  are  so  much  greater  than  of 
ordinary  photography,  the  advice  is  almost  universal  that  no  one  should  try  to 
learn  photography  and  photo-micrography  at  the  same  time,  but  that  one 
should  learn  the  processes  of  photography  by  making  portraits,  landscapes, 
copying  drawings,  etc.,  and  then  when  the  principles  are  learned  one  can  take 
up  the  more  difficult  subject  of  photo-micrography  with  some  hope  of  success. 

The  advice  of  Stern  berg  is  so  pertinent  and  judicious  that  it  is  reproduced  : 


220  PHOTO-MICROGRAPHY  [CH.   VI11 

"  Those  who  have  had  no  experience  in  making  photo-micrographs  are  apt  to 
expect  too  much  and  to  underestimate  the  technical  difficulties.  Objects 
which  under  the  microscope  give  a  beautiful  picture,  which  \ve  desire  to 
reproduce  by  photography  may  be  entirely  unsuited  for  the  purpose.  In 
photographing  with  high  powers  it  is  necessary  that  the  objects  to  be  photo- 
graphed be  in  a  single  plane  and  not  crowded  together  and  overlying  each 
other.  For  this  reason  photographing  bacteria  in  sections  presents  special 
difficulties  and  satisfactory  results  can  only  be  obtained  when  the  sections  are 
extremely  thin  and  the  bacteria  well  stained.  Even  with  the  best  preparations 
of  this  kind  much  care  must  be  taken  in  selecting  a  field  for  photography. 
It  must  be  remembered  that  the  expert  niicroscopist,  in  examining  a  section 
with  high  powers,  has  his  finger  on  the  fine  adjustment  screw  and  focuses  up 
and  down  to  bring  different  planes  into  view.  He  is  in  the  habit  of  fixing  his 
attention  on  the  part  of  the  field  which  is  in  focus  and  discarding  the  rest. 
But  in  a  photograph  the  part  of  the  field  not  in  focus  appears  in  a  prominent 
way  which  mars  the  beauty  of  the  picture." 

APPARATUS    FOR    PHOTO-MICROGRAPHY 

<:  296.  Camera. — For  the  best  results  with  the  least  expenditure  of  time 
one  of  the  cameras  especially  designed  for  photo-micrography  is  desirable  but 
is  not  by  any  means  indispensable  for  doing  good  work.  An  ordinary  photo- 
graphic camera,  especially  the  kind  known  as  a  copying  camera,  will  enable 
one  to  get  good  results,  but  the  trouble  is  increased,  and  the  difficulties  are  so 
great  at  best,  that  one  would  do  well  to  avoid  as  many  as  possible  and  have  as 
good  an  outfit  as  can  be  afforded  (Fig.  170). 

The  first  thing  to  do  is  to  test  the  camera  for  the  coincidence  of  the  plane 
occupied  by  the  sensitive  plate  and  the  ground  glass  or  focusing  screen. 
Cameras  even  from  the  best  makers  are  not  always  correctly  adjusted.  By 
using  a  straight  edge  of  some  kind,  one  can  measure  the  distance  from  the 
inside  or  ground  side  of  the  focusing  screen  to  the  surface  of  the  frame.  This 
should  be  done  all  around  to  see  if  the  focusing  screen  is  equally  distant  at  all 
points  from  the  surface  of  the  frame.  If  it  is  not  it  should  be  made  so.  When 
the  focusing  screen  has  been  examined,  an  old  plate,  but  one  that  is  perfectly 
flat,  should  be  put  into  the  plate  holder  and  the  slide  pulled  out  and  the  dis- 
tance from  the  surface  of  the  plate  holder  determined  exactly  as  for  the  focus- 
ing screen.  If  the  distance  is  not  the  same  the  position  of  the  focusing  screen 
must  be  changed  to  correspond  with  that  of  the  glass  in  the  plate  holder,  for 
unless  the  sensitive  surface  occupies  exactly  the  position  of  the  focusing  screen 
the  picture  will  not  be  sharp,  no  matter  how  accurately  one  may  focus.  In- 
deed, so  necessary  is  the  coincidence  of  the  plane  of  the  focusing  screen  and 
sensitive  surface  that  some  photo-micrographers  put  the  focusing  screen  in 
the  plate  holder,  focus  the  image  and  then  put  the  sensitive  plate  in  the 
holder  and  make  the  exposure  (Cox).  This  would  be  possible  with  the  older 
forms  of  plate  holders,  but  not  with  the  double  plate  holders  mostly  used  at 
the  present  day. 


CH.    VIII'\  PHOTO-MICROCKArilY  221 

2  297.  Size  of  Camera. — The  majority  of  photo -micrographs  do  not 
exceed  8  centimeters  in  diameter  and  are  made  on  plates  Sxii,  10x13  or  13x18 
centimeters  (3'|x4 '4  in.,  4x5  in.,  or  5x7  in.).  Most  of  the  vertical  cameras 
are  for  plates  not  exceeding  10x13  centimeters  (4x5  in.)  but  Zeiss'  new  form 
will  take  plates  21x21  centimeters  (S'^xS1^  in.). 

2  298.  Work  Room. — It  is  almost  self-evident  that  the  camera  must  be  in 
some  place  free  from  vibration.  A  basement  room  where  the  camera  table 
may  rest  directly  on  the  cement  floor  or  on  a  pier  is  excellent.  Such  a  place 
is  almost  necessary  for  the  best  work  with  high  powers.  For  those  living  in 
cities,  a  time  must  also  be  chosen  when  there  are  no  heavy  vehicles  moving  in 
the  streets.  For  less  difficult  work  an  ordinary  room  in  a  quiet  part  of  the 
house  or  laboratory  building  will  suffice. 

2  299.  Arrangement  and  Position  of  the  Camera  and  the  Microscope. — 
For  much  photo-micrography  a  vertical  camera  and  microscope  are  to  be  pre- 
ferred (Fig.  170).  Excellent  arrangements  were  perfected  long  ago,  especially 
by  the  French.  (See  Moitessier.) 

Vertical  photo-micrographic  cameras  are  now  commonly  made,  and  by 
some  firms  only  vertical  cameras  are  produced.  They  are  exceedingly  con- 
venient, and  do  not  require  so  great  a  disarrangement  of  the  microscope  to 
make  the  picture  as  do  the  horizontal  ones.  The  variation  in  size  of  the  pic- 
ture in  this  case  is  mostly  obtained  by  the  objective  and  the  projection  ocular 
rather  than  by  length  of  bellows  (see  below  Fig.  170).  It  must  not  be  forgot- 
ten, however,  that  penetration  varies  inversely  as  the  square  of  the  power,  and 
only  inversely  as  the  numerical  aperture  (  $  40) ,  consequently  there  is  a  real 
advantage  in  using  a  low  power  of  great  aperture  and  a  long  bellows  rather 
than  an  objective  of  higher  power  with  a  short  bellows.  A  horizontal  camera 
is  more  convenient  for  use  with  the  electric  light  also  (Fig.  180) . 

For  convenience  and  rapidity  of  work  a  microscope  with  mechanical  stage 
is  desirable.  It  is  also  an  advantage  to  have  a  tube  of  large  diameter  so  that 
the  field  will  not  be  too  greatly  restricted  (Fig.  176).  In  some  microscopes 
the  tube  is  removable  almost  to  the  nose-piece  to  avoid  interfering  with  the 
size  of  the  image.  The  substage  condenser  should  be  movable  on  a  rack  and 
pinion.  The  microscope  should  have  a  flexible  pillar  for  work  in  a  horizontal 
position.  While  it  is  desirable  in  all  cases  to  have  the  best  and  most  conven- 
ient apparatus  that  is  made,  it  is  not  by  any  means  necessary  for  the  produc- 
tion of  excellent  work.  A  simple  stand  with  flexible  pillar  and  good  fine 
adjustment  will  answer. 

\  300.  Objectives  and  Oculars  for  Photo-Micrography. — The  belief  is 
almost  universal  that  the  apochromatic  objectives  are  most  satisfactory  for 
photography.  They  are  employed  for  this  purpose  with  a  special  projection 
ocular.  Two  low  powers  are  used  without  any  ocular  (Fig.  183).  Some  of  the 
best  work  that  has  ever  been  done,  however,  was  done  with  achromatic  objec- 
tives (work  of  Woodward  and  others).  One  need  not  desist  from  undertaking 
photo-micrography  if  he  has  good  achromatic  objectives.  From  a  somewhat 
extended  series  of  experiments  with  the  objectives  of  many  makers  the  good 


PHOTO-MICROGRAPH} 


[CH. 


modern  achromatic  objectives  were  found  to  give  excellent  results  when  used 
without  an  ocular.  Most  of  them  also  gave  good  results  with  projection 
oculars.  It  must  be  said  however,  that  the  best  results  were  obtained  with  the 
apochromatic  objectives  and  projection  oculars.  It  does  not  seem  to  require 


FIG.  171. — Vertical 
photo-micr graphic  cam- 
era, screen  and  small  table 
The  table  is  about  45  cen- 
timeters high  and  in  the 
legs  are  large  screw  eyes  • 
for  leveling  screws.  The 
operator  can  stand  on  the 
floor  and  perform  all  the 
necessary  operations,  and 
in  adjusting  the  micro- 
scope can  sit  on  a  low 
stool. 

The  screen  is  of  zinc 
and  has  two  heavy  lead 
feet  to  hold  it  steady. 
Near  the  lower  left  hand 
corner  of  the  screen  is  an 
aperture  for  the  light  to 
shine  through  upon  the 
mirror.  This  opening  is 
closed  by  a  black  slide 
which  is  just  balanced  so 
that  it  slays  in  any  posi- 
tion. In  making  the  ex- 
posiire  it  is  raised  suffi- 
ciently to  admit  the  light 
to  the  mirror,  but  the 
stage  is  left  in  shadou*. 
This  screen  shades  the 
microscope  and  the  face 
of  the  operator.  (  Trans. 
Amer.  Micr.  Soc.  sooi.) 


so  much  skill  to  get  good  results  with  apochromatics  as  with  achromatic  ob- 
jectives. The  majority  of  photo-micrographers  do  not  use  the  Huygenian 
oculars  in  photography,  although  excellent  results  have  been  obtained  with 
them.  An  amplifier  is  sometimes  used  in  place  of  an  ocular.  Considerable 
experience  is  necessary  in  getting  the  proper  mutual  position  of  objective  and 


('//. 


223 


amplifier.  The  introduction  of  oculars  especially  designed  for  projection,  has 
led  to  the  discarding  of  ordinary  oculars  and  of  amplifiers.  Oculars  restrict 
the  field  very  greatly,  hence  the  necessity  of  using  the  objective  alone  for 
large  specimens.* 


No.  2. 


No.  4. 


FIG.  172.  Projection  Oculars  with  section 
removed  to  shozv  (lie  construction.  Below  arc 
shown  the  upper  end  with  graduated  circle  to 
indicate  the  amount  of  rotation  found  necessary 
to  focus  the  diaphragm  on  the  screen.  No.  2, 
No.  4.  The  numbers  indicate  the  amount  the 
ocular  magnifies  the  image  formed  by  the  ob- 
ject ire  as  with  the  compensation  oculars, (Zeiss' 
Catalog. ) 


£  301.  Difference  of  Visual  and  Actinic  Foci. — Formerly  there  was  much 
difficulty  experienced  in  photo-micrographing  on  account  of  the  difference  in 
actinic  and  visual  foci.  Modern  objectives  are  less  faulty  in  this  respect  and 
the  apochromatics  are  practically  free  from  it.  Since  the  introduction  of 
orthochromatic  or  isochromatic  plates  and,  in  many  cases  the  use  of  colored 
screens,  but  little  trouble  has  arisen  from  differences  in  the  foci.  This  is 
especially  true  when  mono-chromatic  light  and  even  when  petroleum  light  is 
used.  In  case  an  objective  has  its  visual  and  actinic  foci  at  markedly  different 
levels  it  would  be  better  to  discard  it  for  photography  altogether,  for  the  esti- 
mation of  the  proper  position  of  the  sensitive  plate  after  focusing  is  only  guess 
work  and  the  result  is  mere  chance.  If  sharp  pictures  cannot  be  obtained 
with  an  objective  when  petroleum  light  and  orthochromatic  plates  are  used  the 
fault  may  not  rest  with  the  objective  but  with  the  plate  holder  and  focusing 
screen.  They  should  be  very  carefully  tested  to  see  if  there  is  coincidence  in 
position  of  the  focusing  screen  and  the  sensitive  film  as  described  in  \  296. 

ic  302.  Apparatus  for  Lighting. — For  low  power  work  (35  mm.  and  longer 
focus)  and  for  large  objects,  some  form  of  bull's  eye  condenser  is  desirable 
although  fairly  good  work  may  be  done  with  diffused  light  or  lamp-light 
reflected  by  a  mirror.  If  a  bull's  eye  is  used  it  should  be  as  nearly  achromatic 
as  possible.  The  engraving  glass  shown  in  Fig.  175  answers  well  for  large 

A  comparative  study  both  with  projection  oculars,  and  without  an  ocular 
was  made  with  the  achromatic  objective  25  mm.  (I  inch),  18  mm.  ( ^  inch),  5 
mm.  (i  to  |g  inch)  and  2  mm.  ( T'j  inch)  homogeneous  immersion  of  the 
Bausch  &  Lomb  Optical  Co.;  Gundlach Optical  Co. ;  Leitz  ;  Reichert  ;  Winkel, 
Zeiss  and  the  Spencer  Lens  Co.  Good  results  were  obtained  with  all  of  these 
objectives  both  with  and  without  projection  oculars. 


224 


PHO  TO-MICROGRA  PH  > ' 


[C//.    /'/// 


objects.  For  smaller  objects  a  Steinheil  lens  combination  gives  a  more  bril- 
liant light  and  one  also  more  nearly  achromatic.  For  high  power  work  all  are 
agreed  that  nothing  will  take  the  place  of  an  achromatic  condenser.  This  may 
be  simply  an  achromatic  condenser,  but  preferably  it  should  be  an  apochroma- 
tic  condenser.  Whatever  the  form  of  the  condenser  it  should  possess  dia- 


Oculir  lo 


FIG.  173.  Compensation  Oculars  of  Zciss,  ivith  section  removed  to  show 
the  construction.  The  line  A- A  is  at  the  level  of  the  upper  end  of  the  tube\of 
the  rnicroscape  while  B-B  represents  the  lower  focal  points.  Zeiss  recommends 
the  use  of  the  compensation  oculars  if  one  desires  a  greater  magnification  than. 
the  projection  oculars  give. 


^^mum*^^ 

FIG.  174.     Bull's  eye  lens  and  holder.     (Bausch  &  Lomb  Opt.  Co. ) 


CH.    /'///]  PHOTO-MICKOHKArilY  225 

phragms  so  that  the  aperture  of  the  condenser  may  be  varied  depending  upon 
the  aperture  of  the  objective.  For  a  long  time  objectives  have  been  used  as 
achromatic  condensers,  and  they  are  very  satisfactory,  although  less  conven- 
ient than  a  special  condenser  whose  aperture  is  great  enough  for  the  highest 
powers  and  capable  of  being  reduced  by  means  of  diaphragms  to  the  capacity 
of  the  lower  objectives.  It  should  also  be  capable  of  accurate  centering 

(!*»)• 

j<  303.  Objects  Suitable  for  Photo-micrographs. — While  almost  any  large 
object  may  be  photographed  well  with  the  ordinary  camera  and  photographic 
objective,  only  a  small  part  of  the  objects  mounted  for  microscopic  study  can 
be  photo-uiicrographed  satisfactorily.  Many  objects  that  can  be  clearly  seen 
by  constant  focusing  with  the  fine  adjustment,  appear  almost  without  detail 
on  the  screen  of  the  photo-micrographic  camera  and  in  the  photo-micrograph. 


FK;.  175.  Engraving  glass  to  serve  as  a  con- 
denser and  for  a  dissecting  lens.  (Bausch  &  Lomb 
Opt.  Co.) 


If  one  examines  a  series  of  photo-micrographs  the  chances  are  that  the 
greater  number  will  be  of  diatoms,  plant  sections  or  preparations  of  insects. 
That  is,  they  are  of  objects  having  sharp  details  and  definite  outlines,  so  that 
contrast  and  definiteness  may  be  readily  obtained  (§'107,  118,  157).  Stained 
microbes  also  furnish  favorable  objects  when  mounted  as  cover-glass  prepara- 
tions, but  these  give  color  images  (§  107,  119)  and  require  a  color  screen 
(I  291)- 

For  success  with  preparations  of  animal  tissue  they  must  approximate  as 
nearly  as  possible  to  the  conditions  more  easily  obtained  with  vegetable  prep- 
arations. That  is,  they  must  be  made  so  thin  and  be  so  prepared  that  the  cell 
outlines  have  something  of  the  definiteness  of  vegetable  tissue.  It  is  useless 
to  expect  to  get  a  clear  photograph  of  a  section  in  which  the  details  are  seen 
with  difficulty  when  studying  it  under  the  microscope  in  the  ordinary  way. 

Many  sections  which  are  unsatisfactory  as  wholes,  may  nevertheless  have 
parts  in  which  the  structural  details  show  with  satisfactory  clearness.  In  such 
a  case  the  part  of  the  section  showing  details  satisfactorily  should  be  sur- 
rounded by  a  delicate  ring  by  means  of  a  marker  (see  Figs.  70,  72).  If  one's 
preparations  have  been  carefully  studied  and  the  special  points  in  them  thus 
indicated,  they  will  be  found  far  more  valuable  both  for  ordinary  demonstra- 
tion and  for  photography.  The  amount  of  time  saved  by  marking  one's  speci- 
mens can  hardly  be  overestimated.  The  most  satisfactory  material  for 
making  the  rings  is  shellac  colored  with  lampblack. 

Ten  years  ago  many  histologic  preparations  could  not  be  satisfactorily 
photographed.  Now  with  improved  section  cutters,  better  staining  and 
mounting  methods,  and  with  the  color  screens  ({j  291)  and  isochromatic  plates 


226  PHOTO-MICROGRAPHY  [  CH.    VIII 

('',.  290)  almost  any  preparation  which  shows  the  elements  clearly  when  look- 
ing into  the  microscope  can  be  satisfactorily  photographed.  Good  photo- 
graphs cannot,  however,  be  obtained  from  poor  preparations. 

\  304.  Light. — The  strongest  light  is  sunlight.  That  has  the  defect  of 
not  always  being  available,  and  of  differing  greatly  in  intensity  from  hour  to 
hour,  day  to  day  and  season  to  season.  The  sun  does  not  shine  in  the  evening 
when  many  workers  find  the  only  opportunity  for  work.  Following  the  sun- 
light the  electric  light  is  the  most  intense  of  the  available  lights.  Then  come 
magnesium,  acetylene,  the  lime  light,  the  gas-glow  or  Wellsbach  light  and 
petroleum  light.  The  last  is  excellent  for  the  majority  of  low  and  moderate 
power  work.  And  even  for  2  mm.  homogeneous  immersion  objectives,  the 
time  of  exposure  is  not  excessive  for  many  specimens  (40  seconds  to  3 
minutes).  This  light  is  cheap  and  easily  obtained.  It  has  the  advantage  of 
being  somewhat  yellow,  and  therefore  in  many  cases  makes  the  use  of  a  color 
screen  unnecessary  if  one  uses  isochromatic  plates. 

A  lamp  with  flat  wick  about  40  mm.  (i|^  in.)  wide  has  been  found  most 
generally  serviceable.  For  large  objects  and  low  powers  the  flame  may  be 
made  large  and  the  face  turned  toward  the  mirror.  This  will  light  a  large 
field.  For  high  powers  the  edge  toward  the  mirror  gives  an  intense  light. 
The  ordinary  glass  chimney  answers  well,  especially  where  a  metal  screen  is 
used  as  shown  in  Fig.  171. 

EXPERIMENTS    IN    PHOTO-MICROGRAPHY 

§  305.  The  following  experiments  are  introduced  to  show 
practically  just  how  one  would  proceed  to  make  photo- micrographs 
with  various  powers,  and  be  reasonably  certain  of  fair  success.  If 
one  consults  prints  or  the  published  figures  made  directly  from 
photo-micrographs  it  will  be  seen  that,  excepting  diatoms  and 
bacteria,  the  magnification  ranges  mostly  between  10  and  150 
diameters. 

§  306.  Focusing  in  Photo-Micrography. — For  rough  focus- 
ing and  as  a  guide  for  the  proper  arrangement  of  the  object  one 
uses  a  ground-glass  screen  as  in  gross  photography.  With  the 
ground-glass  screen  one  can  judge  of  the  brilliancy  and  evenness  of 
the  illumination  more  accurately  than  in  any  other  way.  For  final 
and  exact  focusing  two  principal  methods  are  employed  : 

(A).  A  focusing  glass  is  used  either  with  a  clear  screen  or  in  a 
board  screen  as  described  above  (§  282).  The  latter  method  is  like 
focusing  with  the  compound  microscope  and  a  positive  ocular.  If 
the  focusing  glass  is  set  properly  the  focus  should  be  easily  and 
accurately  determined. 


en.  r///] 


PHOTO-MICROGRAPHY 


227 


FIG.  176.  Zeiss'  special  photo-micrographic  stand.  This  is  the  parent 
form  of  pholomicrographic  stands  zvith  large  tube  (T),  handle  in  the 
pillar  and  a  special  fine  adjustment  at  the  side  (W).  At  the  top  is  half  of  the 
light  excluding  sleeve.  (Zeiss'  Catalog). 


228  PHOTO-MICROGRAPHY  [CH.    /'/// 

(B) .  To  enable  the  operator  by  looking  directly  into  the  micro- 
scope to  focus  correctly  for  any  distance  of  the  photographic  plate 
(length  of  bellows),  Foot  and  Strobell  introduced  the  use  of  concave 
spectacle  lenses  ranging  from — r  D  to — 10  D.  ( — i  to — 10  diopters). 

They  have  produced  some  of  the  best  photo-micrographs  of 
recent  years  by  their  method.  (See  for  the  full  account,  Zeit.  wiss. 
Mikroskopie,  Bd.  18,  pp.  421-426  ;  Jour.  Ap.  Microscopy,  Vol.  V. 
1902,  p.  2082). 

In  whatever  way  one  focuses  for  photo-micrography  a  difficulty 
often  appears.  No  matter  how  perfect  the  focus  of  the  microscope 
the  picture  may  be  out  of  focus.  This  may  be  due  to  either  of  two 
things  :  (i)  the  focusing  screen  or  focusing  glass  may  not  be  in  the 
right  position  to  make  the  image  sharp  on  the  sensitive  plate 
(§  282,  296).  (2)  The  microscope  may  get  out  of  focus  while  the 
picture  is  being  made.  The  reason  for  this  change  may  be  the 
gradual  settling  down  of  the  tube  of  the  microscope.  This  may  be 
a  fault  of  the  fine  or  of  the  coarse  adjustment.  It  is  a  good  plan  to 
focus  the  object  carefully  and  after  10  or  15  minutes  to  see  if  the 
focus  is  still  good.  If  the  microscope  will  not  stay  in  focus  one  can- 
not get  a  good  picture.  In  that  case  it  is  necessary  to  study  the 
apparatus  and  see  which  part  of  the  mechanism  is  at  fault. 

§  3°7-  Photo-micrographs  of  20  to  50  Diameters. — For 
pictures  under  15  or  20  diameters  it  is  better  to  use  the  camera  for 
embryos  with  the  objective  in  the  end  of  the  camera,  and  the  special 
microscope  stand  for  focusing  (Fig.  165). 

For  pictures  at  25  to  50  diameters  one  may  use  the  microscope 
with  a  low  objective,  25  to  35  mm.  equivalent  focus,  and  no  ocular 
(Fig.  180).  The  object  is  placed  on  the  stage  of  the  microscope, 
and  focused  as  in  ordinary  observation.  If  a  vertical  microscope  is 
used  the  light  from  the  petroleum  lamp  or  other  artificial  light,  is 
reflected  upward  by  the  mirror.  It  may  take  some  time  to  get  the 
whole  field  lighted  evenly.  Refer  back  to  §  106  for  directions.  In 
some  cases  it  may  be  advisable  to  discard  the  condenser  and  use  the 
mirror  only.  For  some  purposes  one  will  get  a  better  light  by  plac- 
ing the  bull's  eye  or  other  condenser  between  the  lamp  and  the 
mirror  to  make  the  rays  parallel  or  even  to  make  a  sharp  image  of 
the  lamp  flame  on  the  mirror.  Remember  also  that  in  many  cases 
it  is  necessary  to  have  a  color  screen  between  the  source  of  light  and 
the  object  (§  291). 


CH. 


PHO  TO-M1  CROC  R  API!  > 


229 


For  a  horizontal  camera  it  is  frequently  better  to  swing  the 
mirror  entirely  out  of  the  way  and  allow  the  light  to  enter  the  con- 
denser directly  or  after  traversing  the  bull's  eye  (Fig.  174). 
If  the  object  is  small  an  achromatic  combination  like  a  Steinheil 
magnifier  or  an  engraving  glass  is  excellent  (Fig.  175).  When  the 
light  is  satisfactory  as  seen  through  an  ordinary  ocular,  remove  the 
ocular. 

(A)  Photographing  without  an  Ocular. — After  the  removal  of 
the  ocular  put  in  the  end  of  the  tube  a  lining  of  black  velvet  to 
avoid  reflections.  Connect  the  microscope  with  the  camera,  making 
a  light-tight  joint  and  focus  the  image  on  the  focusing  screen.  One 
may  make  a  light-tight  connection  by  the  use  of  black  velveteen  or 
more  conveniently  by  the  Zeiss'  double  metal  hood  which  slips  over 
the  end  of  the  tube  of  the  microscope,  and  into  which  fits  a  metal 
cylinder  on  the  lower  end  of  the  camera  (Figs.  170,  176).  In 
the  first  figure  the  connection  has  been  made. 


FIG.  177.  Zeiss'  Achromatic  Con- 
denser, c.  s.  c.  s.  Centering  screwsfor 
changing  the  position  of  the  condenser 
and  making  its  axis  continuous  with 
that  of  the  microscope.  A  segment 
of  the  condenser  is  cut  away  to  show 
the  combinations  of  lenses.  For  very 
low  powers  the  upper  lens  is  some- 
times screened  off.  There  is  an  iris 
diaphragm  between  the  middle  and 
lower  combinations.  (Zeiss'  Catalog.} 


,Ex  c 


Exc 


A  B  F  G 

FIG.  178.  A.  Shows  that  the  condenser  is  not  centered.  B.  That  it  is 
centered.  {D-D}  Image  of  diaphragm  formed  by  condenser, 

F.  G.  Shows  that  the  flame  (F  I)  illuminating  the  condenser  is  not  cen- 
tral. In  that  case  the  lamp  or  the  mirror  must  be  changed  in  position  until 
the  image  of  the  flame  is  exactly  central.  (See  also  \  92-93.} 


230 


PHQ  TO-AriCROGRAPHY 


[CH.    VIII 


FIG.  179.  Microscope  of  Voigtlander  &  So/in  with  large  tube  delicate  fine 
adjustment  and  mechanical  stage  suitable  for  photo-micrography.  See  also 
Figs.  79,  84,  .S'p,  95.  (Cut  loaned  by  Voightldnder  &  Sohn. ) 


CH.   VIII}  PHOTO-MICROGRAPHY  231 

It  will  be  necessary  to  focus  down  considerably  to  make  the 
image  clear.  Lengthen  or  shorten  the  bellows  to  make  the  image 
of  the  desired  si/.e,  then  focus  with  the  utmost  care.  In  case  the 
field  is  too  much  restricted  on  account  of  the  tube  of  the  microscope, 
remove  the  draw-tube.  When  all  is  in  readiness  it  is  well  to  wait 
for  three  to  five  minutes  and  then  to  see  if  the  image  is  still  sharply 
focused.  If  it  has  become  out  of  focus  simply  by  standing,  a  sharp 
picture  could  not  be'obtained.  If  it  does  not  remain  in  focus,  some- 
thing is  faulty.  When  the  image  remains  sharp  after  focusiug  make 
the  exposure.  From  20  to  60  seconds  will  usually  be  sufficient  time 
with  medium  plates  and  light  as  described.  If  a  color  screen  is  used 
it  will  require  50-300  seconds,  i,,  e.,  2  to  5  times  as  long,  for  a 
proper  exposure  (§  294). 

B.  Photographing  with  a  Projection  Ocular. — If  the  object  is 
small  enough  to  be  included  in  the  field  of  a  projection  ocular  (Fig. 
172)  use  that  for  making  the  negative  as  follows  :  Swing  the  camera 
around  so  that  it  will  leave  the  microscope  free.  Use  an  ordinary 
ocular,  focus  and  light  the  object,  then  insert  a  projection  ocular  in 
place  of  the  ordinary  one,  and  swing  the  camera  back  over  the 
microscope.  It  is  not  necessary  to  use  an  ordinary  ocular  for  the 
first  focusing,  but  as  its  field  is  larger  it  is  easier  to  find  the  part  to 
be  photographed.  The  first  step  is  then  to  focus  the  diaphragm  of 
the  projection  ocular  sharply  on  the  focusing  screen.  Bring  the 
camera  up  close  to  the  microscope  and  then  screw  out  the  eye-lens 
of  the  ocular  a  short  distance.  Observe  the  circle  of  light  on  the 
focusing  screen  to  see  if  its  edges  are  perfectly  sharp.  If  not,  con- 
tinue to  screw  out  the  eye  lens  until  it  is.  If  it  cannot  be  made 
sharp  by  screwing  it  out  reverse  the  operation.  Unless  the  edge  of 
the  light  circle,  i.  e.,  the  diaphragm  of  the  ocular,  is  sharp,  the  re- 
sulting picture  will  not  be  satisfactory. 

It  should  be  stated  that  for  the  X  2  projection  ocular  the  bellows 
of  the  camera  must  be  extended  about  30  or  40  centimeters  or  the 
diaphragm  cannot  be  satisfactorily  focused  on  the  screen.  The  X  4 
projection  ocular  can  be  focused  with  the  bellows  much  shorter. 
For  either  projection  ocular  the  screen  distance  can  be  extended 
almost  indefinitely. 

When  the  diaphragm  is  sharply  focused  on  the  screen,  the 
microscope  is  focused  as  though  no  ocular  were  present,  that  is,  first 
with  the  unaided  eye  then  with  the  focusing  glass,  The  exposure 


232  PHOTO-MICROGRAPHY  \_CH.   VI11 

is  also  made  in  the  same  way,  although  one  must  have  regard  to 
the  greater  magnification  produced  by  the  projection  ocular  and  in- 
crease the  time  accordingly  ;  thus  when  the  X4  ocular  is  used,  the 
time  should  be  at  least  doubled  over  that  when  no  ocular  is  em- 
ployed. The  time  will  be  still  further  increased  if  a  color  screen  is 
used  (§  294). 

Zeiss  recommends  that  when  the  bellows  have  sufficient  length 
the  lower  projection  oculars  be  used,  but  with  a  short  bellows  the 
higher  ones.  It  is  also  sometimes  desirable  to  limit  the  size  of  the 
field  by  putting  a  smaller  diaphragm  over  the  eye  lens.  This  also 
aids  in  making  the  field  uniformly  sharp. 

§  308.  Determination  of  the  Magnification  of  the  Photo- 
Micrograph. — After  a  successful  negative  has  been  made,  it  is 
desirable  and  important  to  know  the  magnification.  This  is  easily 
determined  by  removing  the  object  and  putting  in  its  place  a  stage 
micrometer.  If  the  distance  between  two  or  more  of  the  lines  of 
the  image  on  the  focusing  screen  is  obtained  with  dividers  and  the 
distance  measured  on  one  of  the  steel  rules,  the  magnification  is 
found  by  dividing  the  size  of  the  image  by  the  known  size  of  the 
object  (§  170).  If  now  the  length  of  the  bellows  from  the  tube  of 
the  microscope  is  noted,  say  on  a  record  table  like  that  in  section 
316,  one  can  get  a  close  approximation  to  the  power  at  some  other 
time  by  using  the  same  optical  combination  and  length  of  bellows. 

For  obtaining  the  magnification  at  which  negatives  are  made  it 
is  a  great  advantage  to  have  one  micrometer  in  half  millimeters 
ruled  with  coarse  lines  for  use  with  the  lower  powers,  and  one  in 
o.i  and  o.oi  millimeter  ruled  with  fine  lines  for  the  higher  powers. 

§  309.  Photo-Micrographs  at  a  Magnification  of  100  to 
150  Diameters. — For  this,  the  simple  arrangements  given  in  the 
preceding  section  will  answer,  but  the  objectives  must  be  of  shorter 
focus,  8  to  3  mm.  It  is  better,  however,  to  use  an  achromatic  con- 
denser instead  of  the  engraving  glass  or  the  Steinheil  lens. 

§  310.  Lighting  for  Photo-Micrography  with  Moderate 
and  High  Powers. — (iooto2,50o  diameters).  No  matter  how 
good  one's  apparatus,  successful  photo-micrographs  cannot  be  made 
unless  the  object  to  be  photographed  is  properly  illuminated.  The 
beginner  can  do  nothing  better  than  to  go  over  with  the  greatest 
care  the  directions  for  centering  the  condenser,  for  centering  the 


CH.    VIII] 


/>HO  TO-MICROGRAPH  > 


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PHO  TO-MICROGKA  PH  } 


.  r/// 


CH.    VIII]  PHOTO-MICROGRAPHY  235 

source  of  illumination,  and  the  discussion  of  the  proper  cone  of  light 
and  lighting  the  whole  field,  as  given  in  §  94,  106.  Then  for  each 
picture  the  photographer  must  take  the  necessary  pains  to  light  the 
object  properly.  An  achromatic  condenser  is  almost  a  necessity 
(S  91).  Whether  a  color-screen  should  be  used  depends  upon 
judgment  and  that  can  be  attained  only  by  experience.  In  the  be- 
ginning one  may  try  without  a  screen,  and  with  different  screens 
and  compare  results. 

A  plan  used  by  many  skilled  workers  is  to  light  the  object  and 
the  field  around  it  well  and  then  to  place  a  metal  diaphragm  of  the 
proper  size  in  the  camera  very  close  to  the  plate  holder.  This  will 
insure  a  clean,  sharp  margin  to  the  picture.  This  metal  diaphragm 
must  be  removed  while  focusing  the  diaphragm  of  the  projection 
ocular,  as  the  diaphragm  opening  is  smaller  than  the  image  of  the 
ocular  diaphragm. 

If  the  young  photo- micrographer  will  be  careful  to  select  for 
his  first  trials,  objects  of  which  really  good  photo-micrographs  have 
already  been  made,  and  then  persists  with  each  one  until  fairly  good 
results  are  attained,  his  progress  will  be  far  more  rapid  than  as  if 
poor  pictures  of  many  different  things  were  made.  He  should,  of 
course,  begin  with  low  magnifications. 

§  311.  Adjusting  the  Objective  for  Cover-Glass. — After 
the  object  is  properly  lighted,  the  objective,  if  adjustable,  must  be 
corrected  for  the  thickness  of  cover.  If  one  knows  the  exact  thick- 
ness of  the  cover  and  the  objective  is  marked  for  different  thick- 
nesses, it  is  easy  to  get  the  adjustment  approximately  correct 
mechanically,  then  the  final  corrections  depend  on  the  skill  and 
judgement  of  the  worker.  It  is  to  be  noted  too  that  if  the  objective 
is  to  be  used  without  a  projection  ocular  the  tube-length  is  practi- 
cally extended  to  the  focusing  screen  and  as  the  effect  of  lengthening 
the  tube  is  the  same  as  thickening  the  cover-glass,  the  adjusting 
collar  must  be  turned  to  a  higher  number  than  the  actual  thickness 
of  the  cover  calls  for  (see  §  113). 

S  312.  Photographing  Without  an  Ocular. — Proceed  ex- 
actly as  described  for  the  lower  power,  but  if  the  objective  is  ad- 
justable make  the  proper  adjustment  for  the  increased  tube-length 
(§113-) 

§  313.     Photographing  with  a  Projection  Ocular. — Proceed 


236  PHOTO-MICROGRAPHY  [CH.    VIII 

as  described  in  §  307  B,  only  in  this  case  the  objective  is  not  to  be 
adjusted  for  the  extra  length  of  bellows.  If  it  is  corrected  for  the 
ordinary  ocular,  the  projection  ocular  then  projects  this  correct 
image  upon  the  focusing  screen. 

§  314.  Photo-Micrographs  at  a  Magnification  of  500  to 
2000  Diameters. — For  this  the  homogeneous  immersion  objective 
is  employed,  and  as  it  requires  a  long  bellows  to  get  the  higher 
magnification  with  the  objective  alone,  it  is  best  to  use  the  pro- 
jection oculars. 

For  this  work  the  directions  given  in  §  307  B  must  be  followed 
with  great  exactness.  The  edge  of  the  petroleum  lamp  flame  is  suf- 
ficient to  fill  the  field  in  most  cases.  With  many  objects  the  time 
required  with  good  lamp  light  is  not  excessive  ;  viz. ,  40  seconds 
to  3  minutes.  The  reason  of  this  is  that  while  the  illumination 
diminishes  directly  as  the  square  of  the  magnification,  it  increases 
with  the  increase  in  the  numerical  aperture,  so  that  the  illuminating 
power  of  the  homogenous  immersion  is  great  in  spite  of  the  great 
magnification  (§  40). 

For  work  with  high  powers  a  stronger  light  than  the  petroleum 
lamp  is  employed  by  those  doing  considerable  photo- micrography. 
Good  work  may  be  done,  however,  with  the  petroleum  lamp. 

It  may  be  well  to  recall  the  statement  made  in  the  beginning, 
that  the  specimen  to  be  photographed  must  be  of  special  excellence 
for  all  powers.  No  one  will  doubt  the  truth  of  the  statement  who 
undertakes  to  make  photo-micrographs  at  a  magnification  of  500  to 
2000  diameters. 

If  one  has  a  complete  outfit  with  electric  arc  light  the  time  re- 
quired for  photographing  objects  is  much  reduced,  i.  e.  ranging  from 
i  to  20  seconds  even  with  the  color  screen.  As  the  light  is  so  in- 
tense with  the  arc  light  it  is  necessary  to  soften  it  greatly  for  focus- 
ing. Several  thicknesses  of  ground  glass  placed  between  the  lamp 
and  the  microscope  will  answer.  These  are  removed  before  taking 
the  negative.  It  is  well  also  to  have  a  water  bath  on  the  optical 
bench  to  absorb  the  heat  rays.  This  should  be  in  position  constant- 
ly (see  Fig.  133,  160). 

§  315.  Use  of  Oculars  in  Photo-Micrography. — There  is 
much  diversity  of  opinion  whether  or  not  the  ordinary  oculars  used 


CH.  run 


PHO  TO-MICROGRAPH  Y 


237 


for  observation  should  be  used  in  photographing.     Excellent  results 
have  been  obtained  with  them  and  also  without  them. 


FIG.  183.  Zeiss'  Apochromatic  Projection  Objective 
of  jo  mm.  equivalent  focus,  for  photo-micrography. 
(Zeiss1  Catalog.} 


is  used  for  visual  observation. 


FIG.  184.  Gordon's  Photo-Micrographic 
Apparatus. — In  this  apparatus  there  is  placed 
over  the  ocular  of  the  microscope  a  tube  contain- 
ing a  projection  lens  which  focuses  the  image 
on  the  sensitive  plate  just  as  the  eye  focuses  the 
image  on  the  retina.  A.  The  tube  bearing 
the  plate  at  the  top.  It  is  about  150  mm.  long. 
B-C.  Photographic  plate  about  40  mm.  square, 
contained  in  a  cap  (C)  on  top  of  the  tube  D. 
Shutter  for  making  the  exposure;  F.  A  flange 
fitting  the  draw-tube  and  supporting  the  camera 
(A);  G.  The  microscope  with  a  metal  block 
which  may  be  clamped  in  position  to  prevent 
the  descent  of  the  body^of  the  microscope  during 
the  exposure;  E.  A  focusing  ocular  of  high 
power  placed  on  the  tube  of  the  microscope  to 
ensure  a  perfect  focus.  If  one  has  perfectly 
normal  eyes  the  focus  with  the  ordinary  ocular 
gives  a  sharp  image. 

With  this  apparatus  the  only  change  needed 
in  the  microscope  is  the  addition  of  the  camera 
(A  )  and  the  clamping  of  the  metal  block  (G). 
Then  the  exposure  may  be  made.  The  use  of  a 
color  screen  and  properly  sensitized  plates  apply 
here  as  with  any  apparatus.  "  One  of  the  chief 
advantages  of  this  extremely  simple  method  of 
photomicrography  is  that  the  performance  of 
the  microscope  is  exactly  the  same  as  when  it 
"  four.  Roy.  Micr.  Soc.,  1905,  p.  651. 


238  PHOTO-MICROGRAPHY  [CH.    VIII 

For  great  magnification  Zeiss  recommends  the  use  of  the  com- 
pensation oculars  with  the  apochromatics. 

The  Zeiss  projection  oculars  may  be  used  with  achromatic  ob- 
jectives of  large  aperture  as  well  as  with  the  apochromatics. 

NEGATIVE     RECORD 

Name  No.  Location 


Camera Date 

Exposure 

• 

Objective Developer 

Ocular Fixer 

Condenser Mag.  X  ._. 


Diaphragm . 


Object  Stained  with. 

Color  Screen 

Plate 

Light  and  Hour 


Remarks 


PHOTOGRAPHING    OPAQUE    OBJECTS    AND    METALLIC   SURFACES 
WITH    A    MICROSCOPE 

All  of  the  objects  considered  in  the  first  part  of  this  chapter  are  opaque 
and  some  of  them  were  to  be  photographed  somewhat  larger  than  natural  size. 
*To  meet  the  needs  of  modern  work,  especially  with  metals  and  alloys  one 
must  be  able  to  examine  and  photograph  prepared  surfaces  at  magnifications 
ranging  from  five  or  ten  to  five  hundred  or  more  diameters. 

\  317.  Microscope  for  Opaque  Objects. — If  one  does  not  need  to  magnify 
more  than  about  100  diameters,  any  good  microscope  will  answer.  For  the 
higher  powers  it  is  far  more  convenient  to  employ  a  special  microscope  for 
metallography  (micro-metalloscope.)  (German,  Metallmikroskop;  French, 
Microscope  pour  1'dtude  des  surfaces  me"talliques  et  des  objets  opaque). 


CH.    /'///] 


PHOTO-MICROGRAPHY 


239 


Such  a  microscope  has  the  following  general  characters:  The  stage  is 
movable  up  and  down  with  rack  and  pinion,  it  is  rotary  and  more  or  less 
mechanical  by  means  of  centering  screws.  With  some  at  least  the  stage  may 
be  removed  entirely.  No  substage  condenser  is  present,  and  a  mirror  is  only 
necessary  for  occasional  transparent  objects.  A  revolving  nose-piece  is  not  so 
good  as  an  objective  changer.  See  Fig.  176. 


A 

FIG.  185  FIG.  1 86 

FIG.   185.     Leitz'  Vertical  Illuminator.     (From  Leitz'  Catalog.) 
FIG.  186.     Zeiss"  Vertical  Illuminator.     (From  Zeiss'  Catalog.) 

\  318.  Illumination  of  Opaque  Objects. —  (A)  for  25  to  100  diameters. 
The  directions  of  Mr.  Walmsley  are  excellent  (Trans.  Amer.  Micr.  Soc.,  1898, 
p.  191).  "Altogether  the  best  light  for  the  purpose  is  diffused  daylight. 
Proper  lighting  is  more  easily  obtained  with  a  vertical  camera.  An  even  illum- 
ination avoiding  deep  shadows  is  preferable  in  most  cases  and  is  more  easily 
attained  with  the  object  in  a  horizontal  position.  For  many  objects  it  is  better 
not  to  use  a  bull's  eye  or  any  form  of  condenser  but  for  others  the  condenser 
ma}-  be  needed,  but  when  the  condenser  is  used  one  must  avoid  too  much  glare. 
The  now  little  used  parabolic  reflector  and  Lieberkuhn  serve  well  in  many 
cases,  but  he  adds  "  the  majority  yield  better  results  under  the  most  simple 
forms  of  illuminanion,"  /.  e. ,  with  the  diffused  light  from  the  window.  This 
has  been  the  experience  of  the  writer  also. 

In  case  diffused  daylight  is  employed  the  camera  should  be  near  a  good 
sized  window,  and  the  object  should  be  somewhat  below  the  window  ledge  so 
that  the  illumination  is  partly  from  above  and  from  the  side.  (This  is  easily 
attained  with  the  small  table  and  vertical  camera  shown  in  Figs.  165,  170,  171). 
The  vertical  illuminator  is  advantageous  for  these  powers  also.  See  (B. ). 

(B)     For  100  to   500  diameters, — For   the   magnification   above   50  it   is 


240  PHOTO-MICROGRAPHY  [CH.    I'll  I 

desirable  and  for  those  above  100  it  is  necessary  to  use  some  form  of  "  vertical 
illuminator,"  that  is  some  arrangement  by  which  the  light  is  reflected  down 
through  the  objective  upon  the  object,  the  objective  acting  as  a  condenser, 
and  from  the  object  back  through  the  objective  and  ocular  to  the  eye  of  the 
observer.  This  is  accomplished  in  two  ways: 

(1)  By  means  of  a  small  speculum-metal  mirror  in  the  tube  of  the  micro- 
scope.    This  is  set  at  an  angle  of  45  degrees  and  the  light  thrown  into  the  tube 
upon   it   is  reflected  straight  down  through  the  objective  upon  the  object. 
The  speculum  metal  being  opaque  cuts  out  apart  of  the  light.     Instead   of  a 
metal  mirror  a  circular  disc  of  glass  is  now  more  frequently  used.     This  allows 
the  major  part  of  the  light  reflected  from  the  object  to  pass  up  through  the 
objective,  to  reach  the  eye. 

(2)  By  means  of  a  small  glass  45  degree  prism  inserted   into  the  side  of 
the  objective  or  of  a  special  adapter.     The  light  is  from  the  side  of  the  micro- 
scope, and  is  reflected  by  the  prism  straight  down  through  the  objective  upon 
the  object  as  before.*     See  Figs.  185-186. 

§  319.  Light  for  the  Vertical  Illuminator. — For  moderate 
powers  one  may  place  the  microscope  in  front  of  a  window,  or  one 
may  use  a  petroleum  or  gas  lamp.  For  the  higher  powers  acetylene 
or  preferably  the  electric  arc  light  is  used.  In  either  case  it  may  be 
necessary  to  soften  the  light  somewhat  either  by  a  color  screen  or 
by  some  ground  glass.  The  light  should  be  concentrated  upon  the 
exposed  end  of  the  prism  or  into  the  hole  leading  to  the  glass  disc. 
Both  the  prism  and  the  disc  should  be  adjustable  for  different  objec- 
tives and  different  specimens.  The  cone  of  light,  especially  with 
the  electric  arc  lamp,  should  be  enclosed  in  a  hollow  metal  or  asbes- 
tos cone  to  avoid  the  glare  in  the  eyes  of  the  operator,  and  it  may 


*The  idea  of  the  vertical  illuminator  apparently  originated  with  Hamilton 
L,.  Smith.  He  used  the  metal  reflector.  Beck  substituted  a  cover-glass  and 
Powell  and  Lealand  a  disc  of  worked  glass;  i.  e.  glass  that  had  been  carefully 
polished  and  leveled  on  the  two  sides.  Carpenter-Dallinger,  pp.  336-338. 

The  use  of  the  prism  with  the  objective  is  due  to  Tolles  (See  Jour.  Roy. 
Micr.  Soc.,  vol.  iii,  1880,  pp.  526,  574). 

In  Zeiss'  catalog  the  prism  form  is  figured.  In  the  catalog  of  Nachetboth 
the  glass  disc  and  the  prism  forms  are  figured. 

For  both  these  devices  uncovered  objects  are  most  successful  or  if  the  object 
is  covered  it  must  be  in  optical  contact  with  the  cover-glass.  Naturally  good 
reflecting  surfaces  like  the  rulings  on  polished  metal  bars  give  most  satisfactory 
images,  hence  this  method  of  illumination  is  especially  adapted  to  micro- 
metallography.  Indeed,  without  some  such  adequate  method  of  illumination 
the  study  of  metals  and  alloys  with  high  powers  would  be  impossible.  So  suc- 
cessful is  it  that  oil  immersion  objectives  may  be  used.  (Carpenter-Dallinger, 
PP-  335-338). 


CIL   /7/7]  PHOTO-MICROGRAPHY  241 

be  necessary  to  soften  the  light  with  ground  glass  before  attempt- 
ing to  focus  and  arrange  the  specimen.  This  ground  glass  would 
in  most  cases  be  removed  before  making  the  exposure  (§  314.) 

With  the  electric  light  and  for  long  exposure  or  observation,  a 
water  bath  to  absorb  the  heat  rays  is  necessary  to  avoid  injuring  the 
lenses. 

As  it  is  somewhat  difficult  to  adjust  the  light  in  a  way  to  give 
the  best  effect,  one  can  see  the  advantage  of  the  adjustment  for 
raising  and  lowering  the  stage.  This  serves  for  all  but  the  finest 
focusing,  and  thus  avoids  moving  the  focusing  tube  enough  to 
throw  the  lighting  out  of  adjustment.  It  might  be  advantageous  to 
have  a  fine  adjustment  on  the  stage  also. 

\  320.  Mounting  of  Objects. — For  observation  only  and  with  low  powers, 
objects  may  be  mounted  either  in  a  liquid  or  dry  as  seems  best.  There  should 
be  a  black  background  for  most  objects,  then  light  will  reach  the  eye  only 
from  the  object.  A  light  background  is  sometimes  desirable,  especially  where 
one  cares  only  for  outlines. 

$  321.  Preparation  of  Metallic  Surfaces.  — In  the  first  place  a  flat  face  is 
obtained  by  grinding  or  filing,  and  then  this  is  polished.  For  polishing,  finer 
and  finer  emery  or  other  polishing  powders  are  used,  (rouge  or  diamantine,  or 
specially  prepared  alumina,  etc).  The  aim  is  to  get  rid  of  scratches  so  that 
the  surface  is  smooth  and  free  from  lines. 

\  322.  Etching.  After  the  surface  is  polished  it  should  be  etched  with 
some  substance.  This  etching  material  corrodes  the  less  resistant  material,  the 
edges  of  crystals,  etc.,  so  that  the  structure  appears  clearly.  For  etching, 
tincture  of  iodine,  nitric  acid  in  various  degrees  of  strength,  hydrochloric  acid, 
etc.,  are  used  or  one  may  use  electricity,  the  metal  being  immersed  in  an  indif- 
ferent liquid.  See  numerous  articles  in  the  Metallographist  for  methods  and 
micrographs. 

After  etching,  the  surface  should  be  washed  well  with  water  to  remove  the 
etcher.  Le  Chatelier  recommends  that  the  etched  surface  when  dry  be  covered 
with  a  very  thin  coating  of  collodion  to  avoid  tarnishing.  The  preparation 
will  then  last  for  several  months  untarnished. 

'',.  323.  Mounting  Preparations  of  Metal. — In  order  to  get  a  satisfactory 
image  the  flat,  polished  and  etched  face  should  be  at  right  angles  to  the  optic 
axis.  For  preliminary  observation  one  can  approximate  this  by  mounting  the 
specimen  on  a  piece  of  bees-wax.  (Behrens).  Very  elaborate  arrangements  of 
the  stage  have  also  been  devised  (Reichert). 

2  324.  Photographing  Opaque  Objects.— The  general  directions  given 
in  £  282  should  be  followed  with  the  necessary  modifications.  The  time  of 
exposure  is  usually  considerably  greater  with  opaque  objects  than  with  trans- 
parent ones.  Very  few  such  objects  can  be  photographed  in  less  than  30 


242  PHOTO-MICROGRAPHY  [CH.    VIII 

seconds,  even  with  daylight.  For  metallic  surfaces  and  magnifications  of  100, 
150,  250  to  500,  with  the  electric  arc  light  as  illuminant  the  time  required  for 
favorable  objects  is  i,  2,  4  and  7  seconds;  with  the  Wellsbach  lamp  the  time  is 
5,  10,  30  and  60  minutes  (Sauveur). 

ENLARGEMENTS  ;    LANTERN   SLIDES  ;    PHOTOGRAPHING 
BACTERIAL    CULTURES 

\  325.  Enlargements.  As  a  low  power  objective  has  greater  depth  of 
focus  or  penetration  than  a  higher  power  ( \  40) ,  it  is  desirable  in  many  cases 
to  make  a  negative  of  an  object  with  considerable  depth  at  a  low  magnifica- 
tion, and  then  to  enlarge  this  picture  to  the  desired  size.  As  a  rule  negatives 
will  not  bear  an  enlargement  of  more  than  five  diameters. 

For  this  work  the  camera  shown  in  Fig.  169  is  excellent,  and  the  special 
microscope  stand  shown  in  this  figure  and  in  Fig.  165  enables  one  to  get 
an  exact  focus. 

One  must  select  an  objective  for  the  enlargement  with  a  field  of  sufficient 
size  to  cover  the  part  of  the  negative  to  be  enlarged.  An  objective  of  60  to 
loo  mm.  focus  will  answer  in  most  cases. 

For  the  illumination  the  camera  can  be  elevated  against  the  sky,  or  artifi- 
cial light  may  be  used.  It  is  not  easy  to  light  so  large  a  surface  evenly  by 
artificial  light. 

(A)  Enlargement  on  Bromide  Paper. — For  this  the  negative  is  put  in 
place  and  by  pulling  out  the  bellows  the  proper  amount,  one  gets  the    right 
magnification.     Focus  now  as  for  any  other  object,  using  the   fine  adjustment 
and  focusing  glass. 

For  great  exactness  one  must  put  a  clear  glass  in  the  plate  holder  and  focus 
on  the  surface  away  from  the  objective.  Then  place  the  bromide  paper  on 
this  clear  glass  and  put  another  over  it  to  hold  it  flat  against  the  first  plate  of 
glass.  The  sensitive  surface  will  then  be  in  the  exact  plane  of  the  focus  and 
the  picture  will  be  sharp. 

For  the  development  and  subsequent  treatment  of  the  paper,  follow  the 
directions  of  the  makers. 

(B)  Enlargement  on  a  Glass  Plate. — One  may  proceed  in  enlarging  as 
for  making  lantern  slides  and  make  a  positive  on  a  glass  plate.     If  it  is  then 
desired  to  get  a  negative  for  printing,   place  this  positive  on  the  microscope 
stand  and  make  a  negative  from  it  as  if  it  were  an  object.     Or  one  may  make 
a  contact  impression  as  is  frequently  done  in  lantern  slide  making.     By  this 
method  one  must  make  three  separate  pictures,  (i)  the  original   photo-micro- 
graphic  negative;  (2)  the  enlarged  positive  from  this;  (3)  a  negative  from  the 
enlarged  positive.     With  this  negative  one   may   print  as  from  the  original 
negative. 

\  326.  Lantern  Slides  from  Negatives. — In  preparing  lantern  slides  from 
photo-micrographic  or  ordinary  negatives  one  may  use  the  contact  method,  or 
the  camera.  With  the  camera  one  can  enlarge  or  reduce  to  suit  the  particular 
case.  The  camera  and  special  microscope  stand  shown  in  Fig.  169  are  admir- 


<•//.   /7//J  PHOTO-MICROGRAPHY  243 

able  for  the-  purpose.  For  lantern  slide  work  a  pfiotographic  objective  is  used 
and  the  cone  for  enlargement  removed.  One  may  put  the  objective  in  the 
front  of  the  camera  or  in  the  middle  segment,  making  use  of  the  little  side 
door. 

'',.  327.  Photographing  Bacterial  Cultures  in  Petri  Dishes. — For  tlie  suc- 
cessful photographing  of  these  cultures  dark  ground  illumination  is  employed 
on  the  principle  stated  in  \  103.  That  is  the  preparation  is  illuminated  with 
rays  so  oblique  that  none  can  enter  the  objective.  These  striking  the  culture 
are  reflected  into  the  objective.  The  clear  gelatin  around  the  growth  or  col- 
onies does  not  reflect  the  light  and  therefore  the  space  between  the  colonies  is 
dark. 

For  supporting  the  Petri  dishes  a  hole  is  made  in  a  front  board  for  the 
camera.  This  hole  is  slightly  larger  than  the  dish.  Over  it  is  then  screwed 
or  nailed  a  rubber  ring  slightly  smaller  than  the  Petri  dish.  This  will  stretch 
and  receive  the  dish,  and  grasp  it  firmly  so  that  it  is  in  no  danger  of  falling 
out  when  put  in  a  vertical  position.  If  the  camera  has  two  divisions  like  the 
one  shown  the  board  with  the  Petri  dish  is  put  in  the  front  of  the  camera,  and 
the  objective  in  the  middle  division  through  the  side  door.  Otherwise  the 
board  holding  the  Petri  dish  must  be  on  a  separate  support. 

The  illumination  is  accomplished  by  the  use  of  two  electric  lamps  with 
conical  shades.  (The  cheap  tin  shades  with  white  enamel  paint  on  the  inside 
are  good).  The  lamps  are  placed  at  the  sides  so  that  a  bright  light  is  thrown 
on  the  culture,  but  at  such  an  angle  that  none  of  it  enters  the  objective 
directly. 

A  piece  of  black  velveteen  is  placed  10  to  20  cm.  beyond  the  culture.  This 
prevents  any  light  from  being  reflected  through  the  clear  gelatin  to  the  objec- 
tive. Unless  some  such  precaution  were  taken  the  background  would  be  gray 
instead  of  black. 

One  may  use  daylight  by  putting  the  culture  in  a  support  just  outside  a 
window,  leaving  the  camera  in  the  room.  The  rays  from  the  sky  are  so 
oblique  that  they  do  not  enter  the  objective.  One  must  use  a  black  non-re- 
flecting background  some  distance  beyond  the  dish  as  in  using  artificial  light 
(Atkinson). 

'"/.  328.  Photographing  Bacterial  Cultures  in  Test-Tubes. — Here  the 
lighting  is  as  in  the  preceding  section,  but  a  great  difficulty  is  found  in  getting 
good  results  from  the  refraction  and  reflections  of  the  curved  surfaces.  To 
overcome  this  one  applies  the  principles  discussed  in  \  157,  and  the  test-tubes 
are  immersed  in  a  bath  of  water  or  water  and  glycerin.  The  bath  must  have 
plane  surfaces.  Behind  it  is  the  black  velvet  screen,  and  the  light  is  in  front 
as  for  the  Petri  dishes.  As  suggested  by  Spitta  it  is  well  to  employ  a  bath 
sufficiently  thick  in  order  that  streak  cultures  may  be  arranged  so  that  the  slop- 
ing surface  will  all  be  in  focus  at  once  by  inclining  the  test-tube. 

REFERENCES    FOR   CHAPTER   VIII 

See  the  works  and  journals  dealing  with  photography. 
For*Photo-Micrography  see  Pringle,   Bousfield,  Neuhauss,  3rd  ed.  Stern- 


244 


PHOTO-MICROGRAPH ) 


[C//.    VIII 


berg,  Francotte,  Spitta  an'd  the  special  catalogs  on  photo-micrography  and 
projection  issued'by  the  great  opticians.  The  Journal  of  the  Royal  Micro- 
scopical Society  and  of  the  Ouekett  Micr.  Club;  Zeit.  wiss.  Mikroskopie;  the 
Trans.  Amer.  Micr.  Soc.;  the  Amer.  Monthly  Micr.  Journal;  the  Journal  of 
Applied  Microscopy. 

For  the  photography  of  metallic  surfaces,  see  the  various  journals  of 
engineering  and;  metallurgy,  but  especially  Sauveur's  journal,  the  Metallo- 
graphist,  begun  in  1898;  Jour.  Roy.  Micr.  Soc. 

See  the  works  on'  photo-micrography  and  photography  for  the  details  of 
lantern  slide  making.  See  for  the  Petri  dishes  and  test-tubes,  Atkinson, 
Botanical  Gazette,  xviii  (1893),  p.  333;  Spitta,  Photo-Micrography  (1899),  P- 26. 

For  photography  with  ultra-violet  light  see  Zeiss  special  catalogs.  Jour- 
nal of  the  Royal  Microscopical  Society,  Zeitschrift  fur  wiss.  Mikroskopie; 
Dr.  August  Kohler,  Zeit.  wiss.  Mikr.  Bd.  xxi,  1904,  pp.  129-165,  273-304;  six 
plates;  Band  24,  1907,  pp.  360-366.  Dr.  H.  C.  Ernst  of  the  Harvard  Medical 
School;  Jour.  Med.  Research  N.  S.  Vol.  9,  1905-6  pp.  463-468,  plates. 

4        3   C 


hill 

0 

1 

1  1  1 

1   1 

.$ 
1    1 

0 
1 

III! 

to 

i           1             ' 

Various  Spectra. — These  spectra  illustrate  some  of  the  points  in  the  dis- 
cussion of  color  screens  (§  291). 

The  Solar  spectrum  shows  that  all  the  wave  lengths  of  light  are  present 
except  for  the  very  narrow  dark  lines  (Fraunhofer  lines,  \  214). 

The  Sodium  spectrum  is  an  example  of  the  spectrum  of  an  incandescent 
gas  ;  it  is  also  an  extreme  example  of  monochromatic  light.  Sodium  light  is 
very  brilliant,  but  the  appearance  of  surrounding  objects  gives  one  a  good  idea 
of  the  changed  appearance  which  the  universe  would  assume  if  illuminated  by 
monochromatic  light. 

The  spectra  of  permanganate  and  methemoglobin  illustrate  well  the  ab- 
sorption spectra  of  colored  substances. 

If  one  were  to  use  permanganate  for  a  color  screen  the  object  photograph- 
ing most  successfully  would  be  one  transmitting  light  in  the  E  region  of  the 
spectrum. 

Methemoglobin  would  answer  well  as  a  color  screen  for  an  object  trans- 
mitting light  at  the  violet  end  of  the  spectrum  and  between  the  lines  DE. 


CHAPTER  IX 


SLIDES  AND  COVER-GLASSES;  MOUNTING;  ISOLATION; 

LABELING  AND  STORING  MICROSCOPIC 

PREPARATIONS;  REAGENTS 


SLIDES    AND    COVER-GLASSES 

\  329.  Slides,  Glass  Slides  or  Slips,  Microscopic  Slides  or  Slips. — 
These  are  strips  of  clear  flat  glass  upon  which  microscopic  specimens  are  usually 
mounted  for  preservation  and  ready  examination.  The  size  that  has  been 
almost  universally  adopted  for  ordinary  preparations  is  25  X  ?6  millimeters  (i 
3  inches).  For  rock  sections,  slides  25  X  45  mm.  or  32  X  32  mm.  are  used; 
for  serial  sections,  slides  25  >  76  mm.,  50  X  ?6  mm.  or  38  X  ?6  mm.  are  used. 
For  special  purposes,  slides  of  the  necessary  size  are  employed  without  regard 
to  any  conventional  standard. 

Whatever  size  of  slide  is  used,  it  should  be  made  of  clear  glass  and  the 
edges  should  be  groxind.  It  is  altogether  false  economy  to  mount  microscopic 
objects  on  slides  with  unground  edges.  It  is  unsafe  also  as  the  unground  edges 
are  liable  to  wound  the  hands. 


FIG.  187.  d'lass  slide  or  slip  of  the  ordinary  size  for  microscopic  work  (j 
x  i  in. ,  76 .v  25  mm. }.  (Cut  loaned  by  the  Spencer  Lens  Company}. 

Thick  slides  are  preferred  by  many  to  thin  ones.  For  micro-chemical 
work  Dr.  Chamot  recommends  slides  of  half  the  length  of  those  used  in  ordi- 
nary microscopic  work.  From  the  rapidity  with  which  they  are  destroyed,  he 
thinks  the  ground  edges  are  unnecessarily  expensive.  He  adds  further:  "  It 
is  a  great  misfortune  that  the  colorless  glass  slips  used  in  America  and  so  excel- 
lent for  ordinary  microscopic  work  should  be  easily  attacked  by  all  liquids; 
even  water  extracts  a  relatively  enormous  amount  of  alkalies  and  alkaline 


246 


SLIDES  AND  COVER-CLASSES 


[CIL  IX 


earths.  The  slips  of  greenish  glass,  while  not  as  neat  or  desirable  for  general 
microscopy,  seem  to  be  decidedly  more  resistant,  and  are  therefore  preperable.'' 
Transparent  celluloid  slides  are  recommended  by  Behrens  for  work  where  hy- 
drofluoric acid  and  its  derivaties  are  to  be  examined.  (Chaniot,  Jour,  Appl. 
Micr.  vol.  iii,  p.  793) . 

\  330.  Cleaning  Slides  for  Ordinary  Use. — Place  new  slides  that  are  to  be 
wiped  at  one  sitting  in  a  glass  vessel  of  distilled  water  containing  5°,,  ammonia 
(Fig.  188-189).  F°r  wiping  the  slides  use  a  so-called  glass  towel  or  other  well 
washed  linen  towel.  One  may  avoid  large  wash  bills  by  using  absorbent  gauze.* 

In  handling  the  slides  grasp  them  by  the  edges.  Cover  the  fingers 
of  the  right  hand  with  the  wiping  towel  or  the  gauze  and  rub  both  faces 
with  it.  When  wiped  thoroughly  dry,  place  the  slide  in  a  dry  glass  jar 
like  that  shown  iu  Fig.  189,  or  for  larger  numbers  use  a  museum  jar  (Fig. 
190).  Soap  and  water  are  also  recommended  for  new  slides. 

\  331.  Cleaning  Used  Slides. — If  only  watery  substances  or  glycerin  or 
glycerin  jelly  have  been  used  one  may  soak  the  slides  over  night  in  ammonia 
water,  then  changing  the  water  for  fresh  and  wiping  as  described  iu  ?  330. 

When  balsam  or  other  resinous  media  ($  353)  have  been  used  it  is  best  to 


c 


FIG.  188.  Round  glass  aquarium  jar  suited  for  an 
aquariutn,  for  cleaning  slides  or  for  any  other  purpose 
where  a  wide  open  glass  dish  is  needed. 


FIG.  189.  Covered  glass  dish  knozun  as  an  '•'ointment 
jar"  of  the  right  height  to  hold  slides  on  end.  (Cuts 
j./6,  147  loaned  by  the  Whitall  Tatum  Co. ) . 


*  The  gauze  mentioned  is  No.  10,  "Sterilized  absorbent  gauze",  of  the 
Griswoldville  Mf'g  Co.  of  N.Y.  It  is  sometimes  called  bleached  cheese  cloth. 
In  the  author's  laboratory  it  is  cut  into  pieces,  14,  /i,  y5  of  a  yard.  When  a 
piece  is  soiled  it  is  thrown  away. 


CH.  /A']  SLIDES  ./AY;  ('Ol'EA'-(if.ASS/-:S  247 

heat  the  slides  over  a  Bunsen  flame  and  remove  the  cover-glass.  Place  the 
cover  in  cleaning  mixture  (g  339).  The  slide  may  also  be  placed  in  cleaning 
mixture  or  in  some  hot  water  containing  io"()  gold  dust  or  other  strong  alka- 
line cleaner.  When  the  metal  basin — preferably  an  agate  ware  basin — is  two 
thirds  full  of  the  slides,  heat  until  the  water  conies  to  a  boil.  Then  let  it  cool. 
Add  fresh  water  and  most  of  the  slides  may  be  wiped  clean. 


FIG.  190.  Museum  jar  with  clamp  top 
for  storing  cleaned  slides  and  for  preserving 
specimens.  ( Cut  loaned  by  the  Whitall  Ta- 
tinn  Co.) 


If  dichromate  cleaning  mixture  is  used  the  best  method  is  to  have  a 
museum  jar  of  it  and  drop  the  slides  in  as  they  are  rejected,  or  a  large  number 
at  once  as  is  most  convenient.  It  may  require  a  weeif  or  more  to  clean  the 
slides  with  cleaning  mixture.  As  this  is  a  very  corrosive  mixture  for  metals 
use  onlv  glass  dishes  in  dipping  into  it.  When  the  slides  are  freed  from  balsam 
etc.  pour  off  the  cleaning  mixture  into  another  glass  vessel  and  allow  a  stream 
of  water  to  flow  over  the  slides  until  all  the  cleaning  mixture  has  been  washed 
away.  Then  add  distilled  water  and  wipe  the  slides  from  that.  Any  slides 
still  not  freed  from  the  balsam  should  be  put  back  into  the  cleaning  mixture. 
Apparently  the  slides  are  not  injured  by  a  prolonged  stay  in  the  mixture. 

•ij  332.  Cleaning  Slides  for  Special  Uses. — In  making  blood  films,  for 
micro-chemistry  and  whenever  an  even  film  is  desired  every  particle  of  oily 
substance  must  be  removed.  The  slides  should  be  placed  in  the  dichromate 
cleaning  mixture  ( §  329)  one  day  or  more,  thoroughly  washed  with  clean 
water  and  then  in  distilled  water,  or  in  50%  to  75%  alcohol.  They  are  taken 
from  the  water  or  alcohol  and  wiped  dry  as  needed.  In  wiping  keep  two  or 
more  layers  of  the  absorbent  gauze  over  the  fingers.  Only  one  slide  is  wiped 
with  each  piece  of  gau/.e.  The  surface  to  touch  the  slides  should  never  have 


248  SLIDES  AND  COVER-GLASSES  \_CH.1X 

been  touched  by  the  hands  for  a  minute  amount  of  oily  substance  leaves  a 
stratum  on  the  slide  which  causes  the  liquids  used  to  heap  up  instead  of  flow- 
ing out  perfectly  flat.  That  is,  the  slide  is  wet  with  difficulty  and  the  liquid 
instead  of  forming  a  film  tends  to  assume  the  spheroidal  state.  Sometimes 
new  gauze  or  other  cloth  used  may  not  be  wholly  free  from  oily  substance,  or 
the  soap  was  not  wholly  eliminated  in  washing.  Such  wiping  cloths  will  not 
make  the  slides  ready  for  good  films.  Some  workers  soak  the  gau^e  in  sulfuric 
ether  to  remove  the  last  traces  of  oily  substance.  This  is  done  more  especially 
in  cleaning  cover-glasses  for  films,  see  below.  Burnett,  p.  22,  in  speaking  of 
blood  smears  says  :  "The  slides  should  be  thoroughly  clean.  Unused  slides 
may  be  cleaned  in  strong  soap  or  "gold  dust"  solution,  well  rinsed  in  water, 
then  placed  in  alcohol  from  which  they  are  wiped  and  polished." 

\  333.  Cover-Glasses  or  Covering  Glasses.- — These  are  circular  or  quad- 
rangular pieces  of  thin  glass  used  for  covering  and  protecting  microscopic 
objects.  They  should  be  very  thin,  o.  loto  0.25  millimeter  (see  table,  ?  32-34). 
It  is  better  never  to  use  a  cover-glass  over  0.20  mm.  thick,  then  the  prepara- 
tion may  be  studied  with  a  2  mm.  oil  immersion  as  well  as  with  lower  objec- 
tives. Except  for  objects  wholly  unsuited  for  high  powers,  it  is  a  great  mis- 
take to  use  cover-glasses  thicker  than  the  working  distance  of  a  homogeneous 
objective  (\  69).  Indeed,  if  one  wishes  to  employ  high  powers,  the  thicker 
the  section  the  thinner  should  be  the  cover-glass  (see  $  337). 

The  cover-glass  should  always  be  considerably  larger  than  t/ie  object  over 
which  it  is  placed. 


FIGS.  191-192.  Figures  of  square  and 
of  circular  cover-glasses.  (Cuts  loaned  by 
the  Spencer  Lens  Co. ) 


\  334.  Cleaning  Cover-Glasses  for  Ordinary  Use. — Covers  may  be  cleaned 
well  by  placing  them  in  82%  or  95%  alcohol  containing  hydrochloric  acid  one 
per  cent.  They  may  be  wiped  almost  immediately. 

Remove  a  cover  from  the  alcohol,  grasping  by  the  edge  with  the  left 
thumb  and  index.  Cover  the  right  thumb  and  index  with  some  clean  gauze 
or  other  absorbent  cloth;  grasp  the  cover  between  the  thumb  and  index 
and  rub  the  surfaces  keeping  the  thumb  and  index  well  opposed  on  directly 
opposite  faces  of  the  cover  so  that  no  strain  will  come  on  it,  otherwise  the 
cover  is  liable  to  be  broken. 

When  a  cover  is  dry  hold  it  up  and  look  through  it  toward  soine  dark 
object.  The  cover  will  be  seen  partly  by  transmitted  and  partly  by  reflected 
light,  and  any  cloudiness  will  be  easily  detected.  If  the  cover  does  not  look 
clear,  breathe  on  the  faces  and  wipe  again.  If  it  is  not  possible  to  get  a  cover 
clean  in  this  way  it  should  be  put  again  into  the  cleaning  mixture. 

As  the  covers  are  wiped  put  them  in   a  clean  glass  box  or  Petri  dish. 


CH.  AY] 


SLIDES  A.\D 


'  (.L.ISSES 


249 


I  Iimdle  them  always  by  their  edges,  or  use  fine  forceps.     Do  not  put  the  fingers 
on  the  faces  of  the  covers,  for  that  will  surely  cloud  them. 

'<  \35-  Cleaning  Cover-Glasses  for  Special  Uses. — As  with  slides,  covers 
intended  for  films  or  other  purposes  where  the  last  particles  of  oily  substance 
must  l>e  removed,  are  best  put  one  by  one  into  dichromate  cleaning  mixture 
(i  v-,9'.  After  a  day  or  more  this  is  poured  off  and  a  stream  of  fresh  water 
allowed  to  run  on  the  covers  until  all  the  cleaning  mixture  is  removed.  Then 
distilled  water  is  added  and  allowed  to  stand  a  few  minutes.  This  is  poured 
off  and  S2"n  or  95°,,  alcohol  added.  The  covers  remain  in  this  until  needed. 
In  wiping  use  the  precautions  given  with  slides  (\  332). 


FiGS.  193-194.     Glass  bo.v  and  fetri  dish  for  clean 
coi'cr-glasses.     (Cuts  loaned  by  the  Whitall  Tatuin  Co.). 


Cleaning  Large  Cover-Glasses. — For  serial  sections  and  especially 
large  sections,  large  quadrangular  covers  are  used.  These  are  to  be  put  one 
by  one  into  a  cleaning  mixture  as  for  the  smaller  covers  and  treated  in  every 
way  the  same.  In  wiping  them  one  may  proceed  as  for  the  small  covers,  but 
special  care  is  necessary  to  avoid  breaking  them.  It  is  desirable  that  these 
arge  covers  should  be  thin — not  over  0.15-0.20  mm.  otherwise  high  objectives 
cannot  be  used  in  studying  the  preparations. 


l-'ic..  195.  Micrometer  Calipers  (Brown  and  Sharpe).  Pocket  Calipers, 
graduated  in  inches  or  millimeters,  and  well  adapted  for  measuring  cover- 
glasses. 

i  337.  Measuring  the  Thickness  of  Cover-Glasses. — It  is  of  great  advan- 
tage to  know  the  exact  thickness  of  the  cover-glass  on  an  object;  for,  (a)  in 
studying  the  preparation  one  would  not  try  to  use  objectives  of  a  shorter  work- 
ing distance  than  the  thickness  of  the  cover  ( £  69);  (b)  In  using  adjustable 
objectives  with  the  collar  graduated  for  different  thicknesses  of  cover,  the 


250 


SLIDES  AND  COVER-GLASSES 


[C//.  JX 


collar  can  be  set  at  a  favorable  point  without  loss  of  time;  (c )  For  unadjustable 
objectives  the  thickness  of  cover  may  be  selected  corresponding  to  that  for 
which  the  objective  was  corrected  (see  table,  2  33).  Furthermore,  if  there  is 
a  variation  from  the  standard,  one  may  remedy  it,  in  part  at  least,  by  lengthen- 
ing the  tube  if  the  cover  is  thinner,  and  shortening  it  if  the  cover  is  thicker 
than  the  standard  (§  113). 

Among  the  so  called  No.  i  cover-glasses  of  the  dealers  in  microscopical 
supplies,  the  writer  has  foxmd  covers  varying  from  o.io  mm.  to  0.35  mm.  To 
use  cover-glasses  of  so  wide  a  variation  in  thickness  without  knowing  whether 
one  has  a  thick  or  thin  one  is  simply  to  ignore  the  fundamental  principles,  by 
which  correct  microscopic  images  are  obtained. 

It  is  then  strongly  recommended  that  every  preparation  shall  be  covered 
with  a  cover-glass  whose  thickness  is  known,  and  that  this  thickness  be  indi- 
cated in  some  way  on  the  preparation. 

2  338.  Cover-Glass  Measures,  Testers  or  Gauges. — For  the  purpose  of 
measuring  cover-glasses  there  are  two  very  excellent  pieces  of  apparatus. 
The  micrometer  calipers  (Fig.  195)  used  chiefly  in  the  mechanic  arts,  are  con- 
venient and  from  their  size  are  easily  carried  in  the  pocket.  The  cover-glass 
measurer  specially  designed  for  the  purpose  is  shown  in  Fig.  196  by  which 
covers  may  be  more  rapidly  measured  than  with  the  calipers. 


FIG.  196.  Zeiss'  Cover-Glass 
JMeasurer.  With  this  the 
knife  edge  jaics  are  opened  by 
means  of  a  lever  and  the  cover 
inserted.  The  thickness  may 
then  be  read  off  on  the  face  as 
the  pointer  indicates  the  thick- 
ness in  hundredths  millimeter 
iu  the  outer  circle  and  in  thous- 
andths inch  on  the  inner  circle- 


With  these  measures  or  gauges  one  should  be  certain  that  the  index  stands 
at  zero  when  at  rest.  If  the  index  does  not  stand  at  zero  it  should  be  adjusted 
to  that  point,  otherwise  the  readings  will  not  be  correct. 

As  the  covers  are  measured,  the  different  thicknesses  should  be  put  into 
different  glass  boxes  and  properly  labeled.  Unless  one  is  striving  for  the  most 
accurate  possible  results,  cover-glasses  not  varying  more  than  0.06  mm.  maybe 
put  in  the  same  box.  For  example,  if  one  takes  0.15  mm.  as  a  standard,  covers 
varying  0.03  mm.  on  each  side  may  be  put  into  the  same  box.  In  this  case 
the  box  would  contain  covers  of  o.  12,  0.13,  0.14,  0.15,  0.16,  o  17  and  o.iS  mm. 

\  339.  Bichromate  Cleaning  Mixture  for  Glass. — The  cleaning  mixture 
used  for  cleaning  slides  and  cover-glasses  is  that  commonly  used  in  chemical 
laboratories  :  (Dr.  G.  C.  Caldwell's  Laboratory  Guide  in  Chemistry). 


I'll.  AY]  MOUNTING  PREPARATIONS  251 

Bichromate  of  potash  (K.,Cr,O7)  200  grams 

Water,  distilled  or  ordinary  800  cc. 

Sulphuric  acid  (II, So,)  1200  cc. 

Dissolve  the  dichromate  in  the  water  by  the  aid  of  heat,  using  an  agate  or 
other  metal  dish,  then  pour  it  into  a  heavy  iron  kettle  lined  with  sheet  lead 
(Trans.  Amer.  Micr.  Soc  ,  1899,  p.  107) .  Add  the  sulphuric  acid  to  the 
dissolved  dichromate  in  the  kettle.  The  purpose  of  the  lead  lined  kettle  is  to 
avoid  breakage  from  the  great  heat  developed  upon  the  addition  of  the  sul- 
phuric acid.  The  lead  is  very  slightly  affected  by  the  acid,  iron  would  be 
corroded  by  it. 

For  making  this  mixture,  ordinary  water,  commercial  dichromate  and 
strong  commercial  sulphuric  acid  may  be  used.  It  is  not  necessary  to  employ 
chemically  pure  materials. 

This  is  an  excellent  cleaning  mixture  and  is  practically  odorless.  It  is 
exceedingly  corrosive  and  must  be  kept  in  glass  vessels.  It  may  be  used 
more  than  once,  but  when  the  color  changes  markedly  from  that  seen  in  the 
fresh  mixture  it  should  be  thrown  away.  An  indefinite  sojourn  of  the  slides 
and  covers  in  the  cleaner  does  not  seem  to  injure  them. 

MOUNTING,      AND      PERMANENT       PREPARATION      OF      MICROSCOPIC 

OBJECTS 

i/  340.  Mounting  a  Microscopic  Object  is  so  arranging  it  upon  some 
suitable  support  (glass  slide)  and  in  some  suitable  mounting  medium  that  it 
may  be  satisfactorily  studied  with  the  microscope. 

The  cover-glass  on  a  permanent  preparation  should  always  be  considerably 
larger  than  the  objeel ;  and  where  several  objects  are  put  under  one  cover-glass 
it  is  false  economy  to  crowd  them  too  closely  together. 

\  341.  Temporary  Mounting. — In  a  great  many  cases  objects  do  not  need 
to  be  preserved  ;  they  are  then  mounted  in  any  way  to  enable  one  best  to 
study  them,  and  after  the  study  the  cover  glass  is  removed,  the  slide  cleaned 
for  future  use.  In  the  study  of  living  objects,  of  course  only  temporary 
preparations  are  possible.  With  amoebae,  white  blood  corpuscles,  and  many 
other  objects  both  animal  and  vegetable,  the  living  phenomena  can  best  be 
studied  by  mounting  them  in  the  natural  medium.  That  is,  for  amoebae,  in 
the  water  in  which  they  are  found  ;  for  the  white  blood  corpuscles,  a  drop  of 
blood  is  used  and,  as  the  blood  soon  coagulates,  they  are  in  the  serum.  Some- 
times it  is  not  easy  or  .convenient  to  get  the  natural  medium,  then  some  liquid 
that  has  been  found  to  serve  in  place  of  the  natural  medium  is  used.  For 
many  things,  water  with  a  little  common  salt  (water  100  cc. ,  common  salt  ,'•. 
gram)  is  employed.  This  is  the  so-called  normal  salt  or  saline  solution.  For 
the  ciliated  cells  from  frogs  and  other  amphibia,  nothing  has  been  found  so 
good  as  human  spittle.  Whatever  is  used,  the  object  is  put  on  the  middle  of 
the  slide  and  a  drop  of  the  mounting  medium  added,  and  then  the  cover-glass. 
The  cover  is  best  put  on  with  fine  forceps,  as  shown  in  Fig.  197.  After  the 


252  MOUNTING  PREPARATIONS  \_CH.IX 

cover  is  in  place,  if  the  preparation  is  to  be  studied  for  some  time,  it  is  better 
to  avoid  currents  and  evaporation  by  painting  a  ring  of  castor  oil  around  the 
cover  in  such  a  way  that  part  of  the  ring  will  be  on  the  slide  and  part  on  the 
cover  (Fig.  210). 

FIG.  197.  To  show  the 
method  of  putting  a  cover- 
glass  upon  a  microscopic 
preparation.  The  cover  is 
grasped  by  one  edge,  the 
opposite  e dg e  is  then 
brought  down  to  the  slide,  and  the  cover  gradually  lowered  upon  the  object. 

\  342.  Permanent  Mounting. — There  are  three  great  methods  of  making 
permanent  microscopic  preparations.  Special  methods  of  procedure  are 
necessary  to  mount  objects  successfully  in  each  of  these  ways.  The  best 
mounting  medium  and  the  best  method  of  mounting  in  a  given  case  can  only 
be  determined  by  experiment.  In  most  cases  some  previous  observer  has 
already  made  the  necessary  experiments  and  furnished  the  desired  information. 

The  three  methods  are  the  following  :  (A)  Dry  or  in  air  ($  343);  (B)  In 
some  medium  miscible  with  water,  as  glycerin  or  glycerin  jelly  ($  348) ;  (C) 
In  some  resinous  medium  like  Canada  Balsam  (\  353). 

£  343.  Mounting  Dry  or  in  Air. — The  object  should  be  thoroughly  dry. 
If  any  moisture  remains  it  is  liable  to  cloud  the  cover-glass,  and  the  specimen 
may  deteriorate.  As  the  specimen  must  be  sealed,  it  is  necessary  to  prepare  a 
cell  slightly  deeper  than  the  object  is  thick.  This  is  to  support  the  cover- 
glass,  and  also  to  prevent  the  running  in  by  capillarity  of  the  sealing  mixture. 

\  344.     Order  of  Procedure  in  Mounting  Objects  Dry  or  in  Air. 

1.  A  cell  of  some  kind  is  prepared.     It  should   be   slight!}*   deeper  than 
the  object  is  thick  (§  346). 

2.  The  object  is  thoroughly  dried  (desiccated)  either  in  dry  air  or  by  the 
aid  of  gentle  heat. 

3.  If  practicable  the  object  is  mounted  on   the   cover-glass  ;  if   not  it   is 
placed  in  the  bottom  of  the  cell. 

4.  The  slide  is  warmed  till  the  cement  forming  the  cell  wall  is  somewhat 
sticky,  or  a  very  thin  coat  of  fresh  cement  is  added  ;  the  cover  is  warmed   and 
put  on  the  cell  and  pressed  down  all  around  till   a   shining  ring   indicates   its 
adherence  (\  347). 

5.  The  cover-glass  is  sealed. 

6.  The  slide  is  labeled. 

7.  The  preparation  is  cataloged  and  safely  stored. 

\  345.  Example  of  Mounting  Dry,  or  in  Air. — Prepare  a  shallow  cell 
and  dry  it  (£  346).  Select  a  clean  cover-glass  slightly  larger  than  the  cell. 
Pour  upon  the  cover  a  drop  of  10%'  solution  of  salycilic  acid  in  95",,  alcohol. 
Let  it  dry  spontaneously.  Warm  the  slide  till  the  cement  ring  or  cell  is  some- 


CH.  IX} 


MO  UN  TIN(,'   PR  /•:  I'ARATH  INS 


253 


what  sticky,  then  warm  the  cover  gently  and  put  it  on  the  cell,  crystals  down. 
Press  on  the  cover  all  around  the  edge  (§  347)  seal,  label  and  catalog. 

A  preparation  of  mammalian  red  blood  corpuscles  may  be  satisfactorily 
made  by  spreading  a  very  thin  layer  of  fresh  blood  on  a  cover  with  the  end  of 
a  slide.  After  it  is  dry,  warm  gently  to  remove  the  last  traces  of  moisture  and 
mount  blood  side  down,  precisely  as  for  the  crystals.  One  can  get  the  blood 
as  directed  for  the  Micro-spcctroscopic  work  (\  232). 


FIG.  198.  Turn-Table  for  sealing  cover-glasses  and  making  shallow 
mounting  cells.  (  Cut  loaned  by  the  Bausch  &  Lomb  Opt.  Co.). 

'},  346.  Preparation  of  Mounting  Cells. — (A)  Thin  cells.  These  are  most 
conveniently  made  of  some  of  the  cements  used  in  microscopy.  Shellac  is 
one  of  the  best  and  most  generally  applicable.  To  prepare  a  shellac  cell  place 
the  slide  on  a  turn-table  (Fig.  198)  and  center  it,  that  is,  get  the  center  of  the 
slide  over  the  center  of  the  turn-table.  Select  a  guide  ring  on  the  turn-table 
which  is  a  little  smaller  than  the  cover-glass  to  be  used,  take  the  brush  from 
the  shellac,  being  sure  that  there  is  not  enough  cement  adhering  to  it  to  drop. 
Whirl  the  turn-table  and  hold  the  bfnsh  lightly  on  the  slide  just  over  the 
guide  ring  selected.  An  even  ring  of  cement  should  result.  If  it  is  uneven, 
the  cement  is  too  thick  or  too  thin,  or  too  much  was  on  the  brush.  After  a 
ring  is  thus  prepared  remove  the  slide  and  allow  the  cement  to  dry  spontane- 
ously, or  heat  the  slide  in  some  way.  Before  the  slide  is  used  for  mounting, 
the  cement  should  be  so  dry  when  it  is  cold  that  it  does  not  dent  when  the 
finger  nail  is  applied  to  it. 

A  cell  of  considerable  depth  may  be  made  with  the  shellac  by  adding 
successive  layers  as  the  previous  one  dries. 

(B)  Deep  Cells  are  sometimes  made  by  building  up  cement  cells,  but  more 
frequently,  paper,  wax,  glass,  hard  rubber,  or  some  metal  is  used  for  the  main 
part  of  the  cell.  Paper  rings,  block  tin  or  lead  rings  are  easily  cut  out  with 
gun  punches.  These  rings  are  fastened  to  the  slide  by  using  some  cement  like 
the  shellac. 


254 


MOUNTING  PREPARA  TIONS 


[C/7.  IX 


\  347.  Sealing  the  Cover-Glass  for  Dry  Objects  Mounted  in  Cells. — When 
an  object  is  mounted  in  a  cell,  the  slide  is  warmed  until  the  cement  is  slightly 
sticky  or  a  very  thin  coat  of  fresh  cement  is  put  on.  The  cover-glass  is  warmed 
slightly  also,  both  to  make  it  stick  to  the  cell  more  easily,  and  to  expel  any  re- 
maining moisture  from  the  object.  When  the  cover  is  put  on,  it  is  pressed 
down  all  around  over  the  cell  until  a  shining  ring  appears,  showing  that  there 
is  an  intimate  contact.  In  doing  this  the  the  convex  part  of  the  fine  forceps 
or  some  other  blunt,  smooth  object;  it  is  also  necessary  to  avoid  pressing  on 
the  cover  except  immediately  over  the  wall  of  the  cell  for  fear  of  breaking  the 
cover.  When  the  cover  is  in  contact  with  the  wall  of  cement  all  around,  the 
slide  should  be  placed  on  the  turn-table  and  carefully  arranged  so  that  the 
cover-glass  and  cell  wall  will  be  concentric  with  the  guide  rings  of  the  turn- 
table. Then  the  turn-table  is  whirled  and  a  ring  of  fresh  cement  it  painted, 
half  on  the  cover  and  half  on  the  cell  wall  (Fig.  210).  If  the  cover-glass  is 
not  in  contact  with  the  cell  wall  at  any  point  and  the  cell  is  shallow,  there  will 
be  great  danger  of  the  fresh  cement  running  into  the  cell  and  injuring  or  spoil- 
ing the  preparation.  When  the  cover-glass  is  properly  sealed,  the  prepara- 
tion is  put  in  a  safe  place  for  the  drying  of  the  cement.  It  is  advisable  to  add 
a  fresh  coat  of  cement  occasionally. 


FIG.  199.  Centering  Card.  A  card  with  stops  for  the  slide  and  circles  in 
the  position  occupied  by  the  center  of  the  slide.  If  the  slide  is  put  upon  such  a 
card  it  is  easy  to  arrange  the  object  so  that  it  will  be  approximately  in  the 
center  of  the  slide.  The  position  of  the  long  cover  used  for  serial  sections  is 
also  shown.  (From  the  Microscope,  December, 


I  348.  Mounting  Objects  in  Media  Miscible  with  Water.— Many  objects 
are  so  greatly  modified  by  drying  that  they  must  be  mounted  in  some  medium 
other  than  air.  In  some  ca'ses  water  with  something  in  solution  is  used. 
Glycerin  of  various  strengths,  and  glycerin  jelly  are  also  much  employed 
All  these  media  keep  the  object  moist  and  therefore  in  a  condition  resembling 
the  natural  one.  The  object  is  usually  and  properly  treated  with  gradually 
increasing  strengths  of  glycerin  or  fixed  by  some  fixing  agent  before  being 
permanently  mounted  in  strong  glycerin  or  either  of  the  other  media. 


ClI.  AV]  MOUNTING  IN  GLYCERIN  255 

In  all  of  these  different  methods,  unless  glycerin  of  increasing  strengths 
has  been  used  to  prepare  the  tissue,  the  fixing  agent  is  washed  away  with 
water  before  the  object  is  finally  and  permanently  mounted  in  either  of  the 
media. 

For  glycerin  jelly  no  cell  is  necessary  unless  the  object  has  a  considerable 
thickness. 

•;  349.     Order  of  Procedure  in  Mounting  Objects  in  Glycerin. 

1.  A  cell  must  be  prepared  on  the  slide  if  the  object  is  of  considerable 
thickness  (2  346)  . 

2.  A  suitably  prepared  object  is  placed  on  the  center  of  a  clean  slide,  and 
if  no  cell  is  required    a   centering  card  is  used   to    facilitate   the   centering 

(Fig.  199). 

3.  A  drop  of  pure  glycerin  is  poured  upon  the  object,  or  if  a  cell  is  used, 
enough  to  fill  the  cell  and  a  little  more. 

4.  In  putting  on  the  cover-glass  it  is  grasped  with  fine  forceps  and  the 
under  side  breathed  on  to  slightly  moisten  it  so  that  the  glycerin  will  adhere, 
then  one  edge  of  the  cover  is  put  on  the  cell  or  slide  and  the  cover  gradually 
lowered  upon  the  object  (Fig.  197).     The  cover  is  then  gently  pressed  down. 
If  a  cell  is  used,  a  fresh  coat  of  cement  is  added  before  mounting. 

FIG.  200.  Slide  and  cover-glass  showing  method 
of  anchoring  a  cover-glass  icith  a  glycerin  prepara- 
tion when  no  cell  is  used.  A  cover-glass  so  anchored 
is  not  liable  to  move  when  the  cover  is  being  sealed 


FIG.  20  1.  Glass  slide  with  cover-glass,  a  drop  of 
reagent  and  a  bit  of  absorbent  paper  to  shore  method 
of  irrigation. 

5.  The  cover-glass  is  sealed. 

6.  The  slide  is  labeled. 

7.  The  preparation  is  cataloged  and  safely  stored. 

*'<  35°-     Order  of  Procedure  in  Mounting  Objects  in  Glycerin  Jelly. 

1.  Unless  the  object  is  quite  thick  no  cell  is  necessary  with  glycerin  jelly. 

2.  A  slide  is  gently  warmed  and  placed  on  the  centering  card  (Fig.    199) 
and  a  drop  of  warmed  glycerin  jelly  is  put  on   its   center.     The   suitably   pre- 
pared object  is  then  arranged  in  the  center  of  the  slide. 

3.  A  drop  of  the  warm  glycerin  jelly  is  then  put  on  the  object,  or  if  a  cell 
is  used  it  is  filled  with  the  medium. 

4.  The  cover-glass  is  grasped  with  fine  forceps,  the  lower  side  breathed  on 
and  then  gradually  lowered  upon  the  object  (Fig.   197)   and   gently    pressed 
down. 

5.  After  mounting,  the  preparation  is  left  fiat  in  some  cool  place  till   the 
glycerin  jelly  sets,  then  the  superfluous  amount  is   scraped   arid   wiped   away 
and  the  cover-glass  sealed  with  shellac  (\  347). 


256 


MOUNTING  IN  GLYCERIN  JELLY 


[C//.  IX 


6.  The  slide  is  labeled. 

7.  The  preparation  is  cataloged  and  safely  stored. 

§  351.  Sealing  the  Cover-Glass  when  no  Cell  is  used. —  (A)  For  glycerin 
mounted  specimens.  The  superfluous  glycerin  is  wiped  away  as  carefully  as 
possible  with  a  moist  cloth,  then  four  minute  drops  of  cement  are  placed  at 
the  edge  of  the  cover  (Fig.  200),  and  allowed  to  harden  for  half  an  hour  or 
more.  These  will  anchor  the  cover-glass,  then  the  preparation  may  be  put  on 
the  turn-table  and  ringed  with  cement  while  whirling  the  turn-table. 


FIG.  202.  A — Simple  form  of  moist  chamber  made  with  a  plate  and  howl. 
B,  bowl  serving  as  a  bell  jar;  P,  plate  containing  the  ivater  and  over  which  the 
bowl  is  inverted ;  S,  slides  on  which  are  mounted  preparations  which  are  to  be 
kept  moist.  These  slides  are  seen  endwise  and  rest  upon  a  bench  made  by 
cementing  short  pieces  of  large  glass  tubing  to  a  strip  of  glass  of  the  desired 
length  and  width. 

B — Two  cover-glasses  (C)  made  eccentric,  so  that  they  may  be  more  easily 
separated  by  grasping  the  projecting  edge. 

C — Slide  (S)  with  projecting  cover-glass  (C).  The  projection  of  the  cover 
enables  one  to  grasp  and  raise  it  without  danger  of  moving  it  on  the  slide  and 
thus  folding  the  substance  under  the  cover.  (From  Proc.  Amer.  Micr.  Soc., 
1891). 

(B)  For  objects  in  glycerin  jelly,  Fat-rants'1  solution  or  a  resinous 
medium.  The  mounting  medium  is  first  allowed  to  harden,  then  the  superfluous 
medium  is  scraped  away  as  much  as  possible  with  a  knife,  and  then  removed 
with  a  cloth  moistened  with  water  for  the  glycerin  jelly  and  Farrants'  solution 
or  with  alcohol,  chloroform  or  turpentine,  etc.,  if  a  resinous  medium  is  used. 
Then  the  slide  is  put  on  a  turn-table  and  a  ring  of  the  shellac  cement  added. 
(C)  Balsam  preparations  may  be  sealed  with  shellac  as  soon  as  they  are  pre- 
pared, but  it  is  better  to  allow  them  to  dry  for  a  few  days.  One  should  never 
use  a  cement  for  sealing  preparations  in  balsam  or  other  resinous  media  if  the 
solvent  of  the  cement  is  also  a  solvent  of  the  balsam,  etc.  Otherwise  the 
cement  will  soften  the  balsam  and  finally  run  in  and  mix  with  it,  and  partly 
or  wholly  ruin  the  preparation.  Shellac  is  an  excellent  cement  for  sealing 
balsam  preparations,  as  it  never  runs  in.  Balsam  preparations  are  rarely  sealed. 


CH.  IX ] 


MOI  NT/N<;  IN  BALSAM 


257 


\  352.  Example  of  Mounting  in  Glycerin  Jelly. — For  this  select  some 
stained  and  isolated  muscular  fibres  or  other  suitably  prepared  objects.  (See 
under  isolation  ?  357).  Arrange  them  on  the  middle  of  a  slide,  using  the  cen- 
tering card,  and  mount  in  glycerin  jelly  as  directed  in  2  350.  Air  bubbles  are 
not  easily  removed  from  glycerin  jelly  preparations,  so  care  should  be  taken 
to  avoid  them. 

\  353.  Mounting  Objects  in  Resinous  Media. — While  the  media  tnisci- 
ble  with  water  offer  many  advantages  for  mounting  animal  and  vegetable  tis- 
sues the  preparations  so  made  are  liable  to  deteriorate.  In  many  cases,  also, 
they  do  not  produce  sufficient  transparency  to  enable  one  to  use  high  enough 
powers  for  the  demonstration  of  minute  details. 

By  using  sufficient  care  almost  any  tissue  may  be  mounted  in  a  resinous 
medium  and  retain  all  its  details  of  structure. 

For  the  successful  mounting  of  au  object  in  a  resinous  medium  it  must  in 
some  way  be  deprived  of  all  water  and  all  liquids  not  miscible  with  the  resi- 
nous mounting  medium.  There  are  two  methods  of  bringing  this  about  :  (A) 
By  drying  or  desiccation  (\  355),  and  (B)  by  successive  displacements  (2  356). 


FlG.  203  FIG.  204 

FIG.  203.  Small  spirit  lamp  modified  into  a  balsam  bottle,  a  glycerin  or 
glycerin-jelly  bottle,  or  a  bottle  for  homogeneous  immersion  liquid.  For  all 
of  these  purposes  it  should  contain  a  glass  rod  as  shown  in  the  figure.  By 
adding  a  small  brush,  it  answers  well  fora  shellac  bottle  also. 

FIG.  204.     Capped  balsam   bottle.     This  form    is   more  satisfactory  than  the 
preceding.     (Cut  loaned  by  the  Whitall  Tatum  Co. ) 

\  354.  Order  of  Procedure  in  Mounting  Objects  in  Resinous  Media  by 
Desiccation  : 

i.  The  object  suitable  for  the  purpose  (fly's  wings,  etc.)  is  thoroughly 
dried  in  dry  air  or  by  gentle  heat. 


258  MOUXTING  IN  KALSAM  [  CH.  IX 

2.  The  object  is  arranged  as  desired  in  the  center  of  a  clean  slide  on  the 
centering  card  (Fig.  199). 

3.  A  drop  of  the  mounting  medium  is  put   directly    upon   the   object   or 
spread  on  a  cover-glass. 

4.  The  cover-glass  is  put  on  the  specimen  with  fine   forceps    (Fig.    197), 
bat  in  no  case  does  one  breathe  on   the   cover   as   when  media  miscible   with 
water  are  used. 

5.  The  cover-glass  is  pressed  down  gently. 

6.  The  slide  is  labeled. 

7.  The  preparation  is  cataloged  and  safely  stored  (£  367). 

\  355.  Example  of  Mounting  in  Balsam  by  Desiccation. — Find  a  fresh 
fly,  or  if  in  winter,  procure  a  dead  one  from  a  window  sill  or  a  spider's  web. 
Remove  the  fly's  wings,  being  especially  careful  to  keep  them  the  dorsal  side 
up.  With  a  camel's  hair  brush  remove  any  dirt  that  may  be  clinging  to  them. 
Place  a  clean  slide  on  the  centering  card,  then  with  fine  forceps  put  the  two 
wings  within  one  of  the  guide  rings.  Leave  one  dorsal  side  up,  turn  the  other 
ventral  side  up.  Spread  some  Canada  balsam  on  the  face  of  the  cover-glass 
and  with  the  fine  forceps  place  the  cover  upon  the  wings  (Fig.  197).  Prob- 
ably some  air-bubbles  will  appear  in  the  preparation,  but  if  the  slide  is  put  in 
a  warm  place  these  will  soon  disappear.  Label,  catalog,  etc. 

\  356.  Mounting  in  Resinous  Media  by  a  Series  of  Displacements. — For 
examples  of  this  see  the  procedure  in  the  paraffin  and  in  the  collodion  methods 
Ch.  X.  The  first  step  in  the  series  is  Dehydration,  that  is,  the  water  is  dis- 
placed by  some  liquid  which  is  miscible  both  with  the  water  and  the  next 
liquid  to  be  used.  Strong  alcohol  (95%  or  stronger)  is  usually  employed  for 
this.  Plenty  of  it  must  be  used  to  displace  the  last  trace  of  water.  The  tissue 
may  be  soaked  in  a  dish  of  the  alcohol,  or  alcohol  from  a  pipette  may  be 
poured  upon  it.  Dehydration  usually  occurs  in  the  thin  objects  to  be  mounted 
in  balsam  in  5  to  15  minutes.  If  a  dish  of  alcohol  is  used  it  must  not  be  used 
too  many  times,  as  it  loses  in  strength. 

The  second  step  is  clearing.  That  is,  some  liquid  which  is  miscible  with 
the  alcohol  and  also  with  the  resinous  medium  is  used.  This  liquid  is  highly 
refractive  in  most  cases,  and  consequently  this  step  is  called  clearing  and  the 
liquid  a  clearer.  The  clearer  displaces  the  alcohol,  and  renders  the  object 
more  or  less  translucent.  In  case  the  water  was  not  all  removed,  a  cloudiness 
will  appear  in  parts  or  over  the  whole  of  the  preparation.  In  this  case  the  prep- 
aration must  be  returned  to  alcohol  to  complete  the  dehydration. 

One  can  tell  when  a  specimen  is  properly  cleared  by  holding  it  over  some 
dark  object.  If  it  is  cleared  it  can  be  seen  only  with  difficulty,  as  but  little 
light  is  reflected  from  it.  If  it  is  held  toward  the  window,  however,  it  will 
appear  translucent. 

The  third  and  final  step  is  the  displacement  of  the  clearer  by  the  resinous 
mounting  medium. 

The  specimen  is  drained  of  clearer  and  allowed  to  stand  for  a  short  time 
till  there  appears  the  first  sign  of  dullness  from  evaporation  of  the  clearer  from 


(  '//.  IX  ]       ISO  LA  riON  OF  HISTOLOCIC  ELEMENTS 


259 


the  surface.  Then  a  drop  of  the  resinous  medium  is  put  on  the  object,  and 
finally  a  cover-glass  is  placed  over  it,  or  a  drop  of  the  mounting  medium  is 
spread  on  the  cover  and  it  is  then  put  on  the  object. 

ISOLATION   OF   HISTOLOGIC    ELEMENTS 

',  357.  Isolation,  General. — For  a  correct  conception  of  the  forms  of  the 
cells  and  fibers  of  the  various  organs  of  the  body,  one  must  see  these  elements 
isolated  and  thus  be  able  to  inspect  them  from  all  sides.  It  frequently  occurs 
also  that  the  isolation  is  not  quite  complete,  and  one  can  see  in  the  clearest 
manner  the  relations  of  the  cells  or  fibers  to  one  another. 

The  chemical  agents  or  solutions  for  isolating  are,  in  general,  the  same  as 
those  used  for  hardening  and  fixing.  But  the  solutions  are  only  about  one- 
tenth  as  strong  as  for  fixing,  and  the  action  is  very  much  shorter,  that  is,  from 
one  or  two  hours  to  as  many  days.  In  the  weak  solution  the  cell  cement  or 
connective  tissue  is  softened  so  that  the  cells  and  fibers  may  be  separated  from 
one  another,  and  at  the  same  time  the  cells  are  preserved.  In  fixing  and  hard- 
ening, on  the  other  hand,  the  cell  cement,  like  the  other  parts  of  the  tissue, 
is  made  firmer.  In  preparing  the  isolating  solutions  it  is  better  to  dilute  the 
fixing  agents  with  normal  salt  solution  than  merely  with  water  (\  399). 


ooo 
ooo 
ooo 
ooo 
ooo 


FIG.  A. 


FIG.  B. 


FIG.  206 


FIG.  205  A.  B.  Preparation  Vials  for  Histology  and  Embryology.  This 
represents  the  two  vials,  natural  size,  that  have  been  found  most  useful.  They 
are  kept  in  blocks  with  holes  of  the  proper  size. 

Fig.  206.     Block  with  holes  for  containing  shell  vials. 


260  ISOLATION  OF  HISTOLOC1C  ELEMENTS      {CH.IX 

\  359.  Example  of  Isolation. — Place  a  piece  of  the  trachea  of  a  very 
ecently  killed  animal,  or  the  roof  of  a  frog's  mouth,  in  formaldehyde  dissocia- 
tor  in  a  shell  vial  or  glass  box.  After  half  an  hour,  up  to  two  or  three  days 
excellent  preparations  of  ciliated  cells  may  be  obtained  by  scraping  the  trachea 
or  roof  of  the  mouth  and  mounting  the  scrapings  on  a  slide.  If  one  proceeds 
after  one  hour,  probably  most  of  the  cells  will  cling  together,  and  in  the  vari- 
ious  clumps  will  appear  cells  on  end  showing  the  cilia  or  the  bases  of  the  cells, 
and  other  clumps  will  show  the  cells  in  profile.  By  tapping  the  cover  gently 
with  a  needle  holder  or  other  light  object  the  cells  will  separate  from  one  another, 
and  many  fully  isolated  cells  will  be  seen. 

>!  358.  Isolation  by  Means  of  Formaldehyde. — Formaldehyde  in  normal 
salt  solution  is  one  of  the  very  best  dissociating  agents  for  brain  tissue  and  all 
the  forms  of  epithelium.  It  is  prepared  as  follows:  2  cc.  of  formal,  (that  is,  a 
40%  solution  of  formaldehyde)  are  mixed  with  1000  cc.  of  normal  salt  solution. 
This  acts  quickly  and  preserves  delicate  structures  like  the  cilia  of  ordinary 
epithelia,  and  also  of  the  endymal  cells  of  the  brain.  It  is  satisfactory  for 
isolating  the  nerve  cells  of  the  brain.  For  the  epithelium  of  the  trachea,  in- 
testines, etc.,  the  action  is  sufficient  in  half  an  hour;  good  preparations  may 
also  be  obtained  any  time  within  two  days  or  more.  The  action  on  nerve  tissue 
of  the  brain  and  myel  or  spinal  cord  is  about  as  rapid. 


FIG.  207-208.  Slender  dish  and  Syra- 
cuse watch  glasses  for  use  in  making- 
isolations  etc.  (Cuts  loaned  by  the 
W hi  tall  Tatum  Co.} 


£  360.  Staining  the  Cells. — Almost  any  stain  may  be  used  for  the  formalin 
dissociated  cells.  For  example,  one  may  use  eosin.  This  may  be  drawn  under 
the  cover  of  the  already  mounted  preparation  (Fig.  201),  or  a  new  preparation 
may  be  made  and  the  scrapings  mixed  with  a  drop  of  eosin  before  putting  on 
the  cover-glass.  It  is  an  advantage  to  study  unstained  preparations,  otherwise 
one  might  obtain  the  erroneous  opinion  that  the  structure  cannot  be  seen  un: 
less  it  is  stained.  The  stain  makes  the  structural  features  somewhat  plainer; 
it  also  accentuates  some  features  and  does  not  affect  others  so  markedly. 
Congo  red  is  excellent  for  most  isolated  cells. 

\  361.  Permanent  Preparations  of  Isolated  Cells. — If  one  desires  to  make 
a  permanent  preparation  of  isolated  cells  it  may  be  done  by  placing  a  drop 
of  glycerin  at  the  edge  of  the  cover  and  allowing  it  to  diffuse  under  the 
cover,  or  the  diffusion  may  be  hurried  by  using  a  piece  of  blotting  paper,  as 
shown  in  Fig.  201.  One  may  also  make  a  new  preparation  by  mixing 
thoroughly  some  of  the  isolated  material  with  congo-glycerin.  After  a  few 
minutes  the  cover-glass  may  be  put  on  and  sealed  (2  351).  If  one  adds 
congo-glycerin  to  a  considerable  amount  of  the  isolated  material  it  may  be 
kept  and  used  at. any  time. 


t7/.  7.V]    LABELING  AND  STORING  PREPARATIONS  261 

\  362.  Isolation  of  Muscular  Fibers. — For  this  the  formal  dissociator  may 
be  used  (*.  358),  but  the  nitric  acid  method  is  more  successful  (£  420).  The 
fresh  muscle  is  placed  in  this  in  a  glass  vessel.  At  the  ordinary  temperature 
of  a  sitting  room  (20  degrees  centigrade)  the  connective  tissue  will  be  so  far 
gelatinized  in  from  one  to  three  days  that  it  is  easy  to  separate  the  fascicles 
and  fibers  either  with  needles  or  by  shaking  in  a  test  tube  or  shell  vial  (Fig. 
205)  with  water.  It  takes  longer  for  some  muscles  to  dissociate  than  others, 
even  at  the  same  temperature,  so  one  must  try  occasionally  to  see  if  the  action 
is  sufficient.  When  it  is,  the  acid  is  poured  off  and  the  muscles  washed  gently 
with  water  to  remove  the  acid.  If  one  is  ready  to  make  the  preparations  at 
once  they  may  be  isolated  and  mounted  in  water.  If  it  is  desired  to  keep  the 
specimen  indefinitely  or  several  days,  the  water  should  be  poured  off  and  2% 
formaldehyde  added.  The  specimens  may  be  mounted  in  glycerin,  glycerin 
jelly  or  balsam.  Glycerin  jelly  is  the  most  satisfactory,  however. 

ARRANGING    AND    MOUNTING    MINUTE    OBJECTS 

*  363.  Minute  objects  like  diatoms  or  the  scalesof  insects  may  be  arranged 
in  geometrical  figures  or  in  some  fanciful  way,  either  for  ornament  or  more 
satisfactory  study.  To  do  this  the  cover-glass  is  placed  over  the  guide.  This 
guide  for  geometrical  figures  may  be  a  net-micrometer  or  aseries  of  concentric 
circles.  In  order  that  the  objects  may  remain  in  place,  however,  they  must  be 
fastened  to  the  cover-glass.  As  an  adhesive  substance,  mucilage  or  liquid 
gelatin  (\  415)  thinned  with  an  equal  volume  of  50%  acetic  acid  answers  well. 
A  very  thin  coating  of  this  is  spread  on  the  cover  with  a  needle,  or  in  some 
other  way  and  allowed  to  dry.  The  objects  are  then  placed  on  the  gelatinized 
side  of  the  cover  and  carefully  got  into  position  with  a  mechanical  finger,  made 
by  fastening  a  cat's  whisker  in  a  needle  holder.  For  most  of  these  objects  a 
simple  microscope  with  stand  (Figs.  149,  164)  will  be  found  of  great  ad  vantage. 
After  the  objects  are  arranged,  one  breathes  very  gently  on  the  cover-glass  to 
soften  the  mucilage  or  gelatin.  It  is  then  allowed  to  dry  and  if  a  suitable 
amount  of  gelatin  has  been  used,  and  it  has  been  properly  moistened,  the  objects 
will  be  found  firmly  anchored.  In  mounting  one  may  use  Canada  balsam  or 
mount  dry  on  a  cell  (\  343,  353).  See  Newcomer,  Amer.  Micr.  Soc.'s  Proc., 
I.SS6,  p.  ii-8;  see  also  E.  H.  Griffith  and  H.  L.  Smith,  Amer.  Jour,  of  Micros., 
iv,  102,  v.Sj;  Amer.  Monthly  Micr.  Jour.,  i,  66.  107,  113.  Cunningham,  The 
Microscope,  viii,  1888,  p.  237. 

LABELING,       CATALOGING      AND      STORING       MICROSCOPIC 
PREPARATIONS 

2  364.  Every  person  possessing  a  microscopic  preparation  is  interested 
in  its  proper  management  ;  but  it  is  especially  to  the  teacher  and  investigator 
that  the  labeling,  cataloging  and  storing  of  microscopic  preparations  are  of 
importance.  "  To  the  investigator,  his  specimens  are  the  most  precious  of  his 
possessions,  for  they  contain  the  facts  which  he  tries  to  interpret,  and  they 


262  LABELING  AND  STORING  PREPARATIONS   [CH.  AY 

remain  the  same  while  his  knowledge,  and  hence  his  power  of  interpretation, 
increase.  They  thus  form  the  basis  of  further  or  more  correct  knowledge  ;  but 
in  order  to  be  safe  guides  for  the  student,  teacher,  or  investigator,  it  seems  to 
the  writer  that  every  preparation  should  possess  two  things  :  viz,  a  label  and 
a  catalog  or  history.  This  catalog  should  indicate  all  that  is  known  of  a  speci- 
men at  the  time  of  its  preparation,  and  all  of  the  processes  by  which  it  is 
treated.  It  is  only  by  the  possession  of  such  a  complete  knowledge  of  the 
entire  history  of  a  preparation  that  one  is  able  to  judge  with  certainty  of  the 
comparative  excellence  of  methods,  and  thus  to  discard  or  improve  those  which 
are  defective.  The  teacher,  as  well  as  the  investigator,  should  have  this  infor- 
mation in  an  accessible  form,  so  that  not  only  he,  but  his  students  can  obtain 
at  any  time,  all  necessary  information  concerning  the  preparations  which  serve 
him  as  illustrations  and  them  as  examples.  " 

\  365.  Labeling  Ordinary  Microscopic  Preparations. — The  label  should 
possess  at  least  the  following  information. 

The  No.  of  the  preparation,  its  name  and  date  and  the  thickness  of  the 
sections  and  of  the  cover-glass. 


C./.T 


DATE. 


FIG.  209.     Example  of  a  label  of  an   ordinary   his- 
tologic  specimen.   (See  also  Fig.  159  for  serial  sections] 


fit 


%  366.  Cataloging  Preparations. — It  is  believed  from  personal  experience, 
and  from  the  experience  of  others,  that  each  preparation  (each  slide  or  each 
series)  should  be  accompanied  by  a  catalog  containing  at  least  the  informa- 
tion suggested  in  the  following  formula.  This  formula  is  very  flexible,  so 
that  the  order  may  be  changed,  and  numbers  not  applicable  in  a  given  case 
may  be  omitted.  With  many  objects,  especially  embryos  and  small  animals, 
the  time  of  fixing  and  hardening  may  be  months  and  even  j'ears  earlier  than 
the  time  of  imbedding.  So,  too,  an  object  may  be  sectioned  a  long  time  after 
it  was  imbedded,  and  finally  the  sections  may  not  be  mounted  at  the  time  they 
are  cut.  It  would  be  well  in  such  cases  to  give  the  date  of  fixing  under  2,  and 
under  5,  6  and  8,  the  dates  at  which  the  operations  were  performed  if  they 
differ  from  the  original  date  and  from  one  another.  In  brief,  the  more  that  is 
known  about  a  preparation  the  greater  its  value. 

%  367.     General  Formula  for  Cataloging  Microscopic  Preparations : 

1.  The  general  name    and    source.     Thickness    of    cover-glass    and    of 
section. 

2.  The  number  of  the  preparation  and  the  date   of   obtaining   and   fixing 
the  specimen  ;  the  name  of  the  preparator. 

3.  The  special  name  of  the  preparation  and   the    common   and   scientific 
name  of  the  object  from  which  it  is  derived.     Purpose  of  the  preparation. 

4.  The  age  and  condition  of  the   object   from   which   the   preparation   is 


CII.  IX}    LA  HE  I. INI,'  AND  STOR1\C  PREPARATIONS  263 

derived.  Condition  of  rest  or  activity  ;  fasting  or  full  fed  at  the  time  of  death. 

5.  The  chemical  treatment, — the  method  of  fixing,  hardening,    dissociat- 
ing, etc.,  and  the  time  required. 

6.  The    mechanical    treatment, — imbedded,    sectioned,     dissected    with 
needles,  etc.     Date  at  which  done. 

7.  The  staining  agent  or  agents  and  the  time  required  for  staining. 

8.  Dehydrating  and  clearing  agent,  mounting  medium,  cement  used   for 
sealing. 

9.  The  objectives  and   other  accessories   (micro-spectroscope,   polarizer, 
etc.,)  for  studying  the  preparation. 

10.  Remarks,  including  references  to  original  papers,  or  to   good   figures 
and  descriptions  in  books. 

2  368.     A  Catalog  Card  Written  According  to  this  Formula  : 
Muscular  Fibers.      Cat. 

C.  0.15  mm. 
Fibers  20  to  40  n  thick. 

2.  No.  475.     (Drr.  IX)  Oct.  i,  1891.     S.  H.  G.,  Preparator. 

3.  Tendinous  and  intra-muscular  terminations  of  striated  muscular   fibers 
from  the  Sartorius  of  the  cat  ( Felis  domestica) . 

4.  Cat  eight  months  old,  healthy  and  well  nourished.     Fasting  and  quiet 
for  12  hours. 

5.  Muscle  pinned  on  cork  with  vaselined  pins  and  placed  in  20  per   cent 
nitric  acid  immediately  after  death  by  chloroform.     Left  36  hours  in  the  acid; 
temperature  20°  C.     In  alum  water  ()4  sat.  aq.  sol.)  i  day. 

6.  Fibers  separated  on  the  slide  with  needles,  Oct.  3. 

7.  Stained  5  minutes  with  Delafield's  hematoxylin. 

8.  Dehydrated  with  95%  alcohol  5  minutes,  cleared  5  minutes  with  carbol- 
turpentine,  mounted  in  xylene  balsam  ;  sealed  with  shellac. 

9.  Use  a  16  mm.  for  the  general  appearance  of  the  fibers,    then   a  2   or   3 
mm.    objective    for    the    details    of    structure.     Try    the    micro-polariscope 
('i  240,  248). 

10.  The  nuclei  or  muscle  corpuscles  are  very  large  and  numerous  ;  many 
of  the  intra-muscular  ends  are  branched.     See  S.  P.  Gage,  Proc.  Amer.    Micr. 
Soc.,  1890,  p.  132;  Ref.  Hand-book  Med.,  Sci.,  Vol.  V.,  p.  59. 

%  369.  General  Remarks  on  Catalogs  and  Labels. — It  is  especially  desir- 
able that  labels  and  catalogs  shall  be  written  with  some  imperishable  ink. 
Some  form  of  water-proof  carbon  ink  is  the  most  available  and  satisfactory. 
The  water-proof  India  ink,  or  the  engrossing  carbon  ink  of  Higgins,  answers 
well.  As  purchased,  the  last  is  too  thick  for  ordinary  writing  and  should  be 
diluted  with  one-third  its  volume  of  water  and  a  few  drops  of  strong  ammonia 
added. 

If  one  has  a  writing  diamond  it  is  a  good  plan  to  write  a  label  with  it  on 
one  end  of  the  slide.  It  is  best  to  have  the  paper  label  also,  as  it  can  be  more 
easily  read. 


264  LABELING  AND  STORING  PREPARATIONS    \_CH.  IX 

The  author  has  found  stiff  cards,  12^2x7^  cm.,  like  those  used  for  catalog- 
ing books  in  public  libraries,  the  most  desirable  form  of  catalog.  A  specimen 
that  is  for  any  cause  discarded  has  its  catalog  card  destroyed  or  stored  apart 
from  the  regular  catalog.  New  cards  may  then  be  added  in  alphabetical  order 
as  the  preparations  are  made.  In  fact  a  catalog  on  cards  has  all  the  flexibility 
and  advantage  of  the  slip  system  of  notes  (See  Wilder  &  Gage,  p.  45). 

Some  workers  prefer  a  book  catalog.  Very  excellent  book  catalogs  have 
been  devised  by  Ailing  and  by  Ward  (  Jour.  Roy.  Micr.  Soc.,  1887,  pp.  173, 
348;  Amer.  Monthly  Micr.  Jour.  ,1890,  p.  91;  Amer.  Micr.  Soc.  Proc.,  1887,  p. 

233)- 

The  fotirth  division  has  been  added  as  there  is  coming  to  be  a  strong  belief, 
practically  amounting  to  a  certainty,  that  there  is  a  different  structural  appear- 
ance in  many  if  not  all  of  the  tissue  .elements  depending  upon  the  age  of  the 
animal,  upon  its  condition  of  rest  or  fatigue;  and  for  the  cells  of  the  digestive 
organs,  whether  the  animal  is  fasting  or  full  fed.  Indeed  as  physiological  his- 
tology is  recognized  as  the  only  true  histology,  there  will  be  an  effort  to  deter- 
mine exact  data  concerning  the  animal  from  which  the  tissues  are  derived. 
(See  Minot,  Proc.  Amer.  Assoc.  Adv.  Science,  1890,  pp.  271-289;  Hodge,  on 
nerve  cells  in  rest  and  fatigue,  Jour.  Morph.,  vol  VII.  (1892),  pp.  95-168;  Jour. 
Physiol.,  vol.  XVII.,  pp.  129-134;  Gage,  The  processes  of  life  revealed  by  the 
microscope;  a  plea  for  physiological  histology,  Proc.  Amer.  Micr.  Soc.,  vol. 
XVII.  (1895),  pp  3-29;  Science,  vol.  II.,  Aug.  23,  1895,  pp.  209-218.  Smith- 
sonian Institution  ;  Report  for  1896,  pp.  381-396. 

CABINET    FOR     MICROSCOPIC    PREPARATIONS 

£  370.  While  it  is  desirable  that  microscopic  preparations  should  be 
properly  labeled  and  cataloged,  it  is  equally  important  that  they  should  be  pro- 
tected from  injury.  During  the  last  few  years  several  forms  of  cabinets  or 
slide  holders  have  been  devised.  Some  are  very  cheap  and  convenient  where 
one  has  but  a  few  slides.  For  a  laboratory  or  for  a  private  collection  where 
the  slides  are  numerous  the  following  characters  seem  to  the  writer  essential  : 

(i).  The  cabinet  should  allow  the  slides  to  lie  flat,  and  exclude  dust  and 
light. 

(2).  Each  slide  or  pair  of  slides  should  be  in  a  separate  compartment. 
At  each  end  of  the  compartment  should  be  a  groove  or  bevel,  so  that  upon 
depressing  either  end  of  the  slide  the  other  may  be  easily  grasped  (Fig.  210). 
,  It  is  also  desirable  to  have  the  floor  of  the  compartment  grooved  so  that  the 
slide  rests  only  on  two  edges,  thus  preventing  soiling  the  slide  opposite  the 
object. 

(3).  Each  compartment  or  each  space  sufficient  to  contain  one  slide  of 
the  standard  size  should  be  numbered,  preferably  at  each  end.  If  the  com- 
partments are  made  of  sufficient  width  to  receive  two  slides,  then  the  double 
slides  so  frequently  used  in  mounting  serial  sections  may  be  put  into  the  cabi- 
net in  any  place  desired. 

(4).     The  drawers  of  the  cabinet  should  be  entirely  independent,  so  that 


('//.  /A']  C.in/.\'ETS  AND  TRAYS  J-VR  PREPARATIONS 


265 


any  drawer  may  be   partly   or   wholly    removed    without    disturbing   any   of 
the  others. 


Fir,.  210.    A  f>art  of  a  cabinet 'drawer 

from  above.  In  compartment  No.  96 
is  represented  a  slide  lying  /fat.  The  label 
of  the  slide  and  the  number  of  the  compart- 
ment are  so  plaeed  that  the  number  of  the 
compartment  may  be  seen  through  the  slide. 
The  sealing  cement  is  removed  at  one  place 
to  shoo.'  that  in  sealing  the  cover-glass,  the 
cement  is  put  partly  on  the  cover  and  partly 
on  the  slide. 


/>'. —  This  represents  a  section  of  the 
same  part  of  the  drawer,  (a)  Slide  resting 
as  in  a.  ATo.  06.  The  preparation  is  seen  to 
be  above  a  groove  in  the  floor  of  the  com- 
partment. (/>)  One  end  of  the  slide  is  seen 
to  be  uplifted  by  depressing  the  other  into 
the  (•: 


96 



0 

M.96  /sso 

Strife  fite,$ 
±& 

70 

FIG.  211.  Cabinet  for 
.  \Iiiroscopic  Sp  e  c  i  m  ens, 
showing  the  method  of  ar- 
rangement and  of  number- 
ing (he  drawers  and  indi- 
cating the  number  of  the 
first  and  last  compartment 
in  each  drawer.  It  is  bel- 
ter to  have  the  slides  on 
which  the  drawers  rest 
somewhat  shorter,  then  the 
drawer  front  may  be  entire 
and  not  notched  as  here 
shown.  (From  Proc.  Amer. 
Micr.  Soc.,  1883.) 


266  CABINETS  AND  TRAYS  FOR  PREPARATIONS  \CH.  IX 


(5).  On  the  front  of  each  drawer  should  be  the  number  of  the  drawer  in 
Roman  numerals,  and  the  number  of  the  first  and  last  compartment  in  the 
drawer  in  Arabic  numerals  (Fig.  211). 


FIG.  212.  Trays  for  slides  and  for  ribbons  of  sections.  The  figures  show 
the  construction.  It  is  important  to  have  the  bordering  frame  with  rounded 
corners  so  that  the  trays  may  be  easily  pulled  out  of  a  pile  or  reinserted.  The 
screw  eye  shown  in  A  makes  it  easy  to  pull  out  a  single  tray.  For  ribbons  of 
sections  a  piece  of  paper  is  placed  in  the  tray  and  the  ribbons  are  placed  on  it. 
(A)  Face  view,  (B)  Sectional  viezv  of  the  whole  tray,  (C)  Sectional  view  of  one 
side  (natural  size)  to  show  the  construction  more  clearly.  These  trays  arc 
about  30  .v  44  centimeters  ( n  3-4  x  ij  1-4  in. ) ,  and  hold  50  /  x  j  /;/.  slides,  i.  e. , 
5  rows  10  in  a  row.  Trays  of  this  kind  are  so  cheap  ($17.50  per  hundred  for 
those  holding  50  to  60  slides),  that  a  laboratory  can  have  all  that  are  needed. 
(Trans.  Amer.  Micr.  Soc.,  1899,  p.  107.) 

§  371.  Trays  for  Slides  and  Ribbons  of  Sections. — Early  in  1897  the 
writer  devised  the  simple  tray  shown  in  Fig.  212.  It  was  designed  especially  for 
the  ribbons  of  sections  in  preparing  embryologic  series  and  for  material  for 
class  work.  As  will  be  seen  by  the  figure  the  two  sides  are  alike  and  the  tray 
is  very  shallow.  It  was  soon  found  that  the  wood  forming  the  bottom,  of  the 
tray  was  too  rough  for  ribbons  of  sections  and  smooth  white  paper  was  put  in 
the  tray  before  the  ribbons  were  laid  upon  it. 

These  trays  were  soon  used  for  the  mounted  preparations  as  well  as  for  the 
ribbons  of  sections.  They  were  made  of  a  proper  size  to  fit  the  laboratory 
lockers  (Fig.  214);  and  naturally  came  to  be  used  for  storage  instead  of  the 
expensive  slide  cabinets  shown  in  Figs.  210-211.  For  this  purpose  five  could 
be  put  in  a  single  compartment  of  the  locker  or  35  in  an  entire  locker.  As 
each  tray  holds  fifty  slides  I  x  3  in;  37,  1^x3  and  25  slides  2x3  in., the  sav- 
ing of  space  was  very  great. 


<•//.  IX}  CABINETS  sL\D  TRAYS  /-'OK  PREPARATIONS 


267 


$  372.  Slide  Trays  with  Tongue  and  Groove. — In  the  first  trays  the  edges 
were  square  and  sharp.  These  were  rounded  in  later  trays,  but  there  still  re- 
mained a  defect,  for  if  one  wished  to  pile  up  five  to  twenty  trays  on  the  table, 
they  would  not  stay  in  an  even  stack.  To  remedy  this  defect  the  long  way  of 
the  frame  was  tongued  on  one  side  and  grooved  on  the  other  as  shown  in  Fig. 
213.  This  is  a  great  improvement  as  one  can  make  even  stacks  of  25  or  50 
trays,  and  they  will  stay  in  position.  Furthermore  it  renders  the  groups  of  5 
trays  stored  in  the  locker  compartments  much  easier  to  manage,  as  one  can  re- 
move any  of  the  five  trays  without  getting  the  others  disarranged  as  so  often 
occured  with  the  old  form,  lacking  tongue  and  groove. 


O 


FiG.  213.  Slide  Tray  zuith  Cross  Pieces  on  one  Face  to  retain  the  Slides  in 
Rows.  {Dr.  Greenman' s  improvement.)  A  tongue  and  groove  serve  to  hold 
the  trays  in  position  ivhen  they  are  piled  up.  (A.  about  1-8,  and  C.  about 
natural  size.)  The  corners  of  the  tray  frame  are  held  in  place  by  the  corru- 
gated pieces  of  iron  used  in  the  construction  of  picture  frames . 

\  373.  Slide  Trays  with  One  Side  Divided. — A  defect  of  the  trays  for 
storage  is  the  ease  with  which  the  slides  get  disarranged  unless  the  tray  is  en- 
tirely full.  To  avoid  this  defect  Dr.  M.  J.  Greenman  of  the  Wistar  Institute 
divides  one  face  into  rows  of  the  right  width  for  receiving  the  slides.  Then 
while  the  slides  in  any  single  row  might  get  displaced  those  of  neighboring 
rows  cannot  become  mixed  (Fig.  213  A. ).  One  side  of  this  tray  is  smooth  and 
can  be  used  for  ribbons  of  sections  like  the  original  tray.  Dr.  Greenman  stores 
the  trays  in  metal  cabinets,  each  tray  having  a  separate  pair  of  "  runs"  as  is 
shown  in  Fig.  211.  The  author  of  this  book  adds  the  cross  pieces  to  divide 
the  tray  into  rows  and  also  has  the  frame  grooved  and  tongued  (J>  372).  Thus 


268 


PREPARATION  OF  REAGENTS 


.  IX 


constructed  the  tray  is  very  reasonable  in  price  and  most  useful  for  the    needs 
of  a  modern  biologic  laboratory.* 


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FIG.  214.     Student  Locker  with  trays  and  reagent  boards. 
(Jour.  Apl.  Micr.  r$98,  p.  127. ) 

PREPARATION    OF    REAGENTS 


2  374.  General  on  Preparation  of  Reagents.— In  preparing  reagents  both 
heights  and  measures  are  used.  As  a  rule  the  amounts  given  are  those  which 
experience  has  shown  to  give  good  results.  Variations  in  the  proportions  of 
the  mixtures  are  sometimes  advantageous,  and  in  almost  every  case  a  slight 
change  in  the  proportions  makes  no  difference.  Most  laboratory  reagents  are 


*  In  Ithaca,  these  trays  are  made  and  furnished  by  the  H.  J.  Bool  Furni- 
ture Co.  The  cost  per  100  of  the  original  form  is  $17.50  (§  371);  for  the  form 
with  tongue  and  groove,  it  is  $22.50  ;  and  for  the  form  with  tongue  and  groove 
and  one  side  divided  into  rows  ( §  373),  the  cost  is  $30  per  hundred. 


PREPARATION 


KEA<;/<:NTS 


269 


like  food,  good  even  under  quite  diverse  proportions  and  methods  of  prepara- 
tion.    With  a  few,  however,  it  is  necessary  to  have  definite  strengths. 


FIGS.  215-217.     Graduates  of  various  forms  for  measuring  liquids. 
(Cuts  loaned  by  the  Whitall  To  turn  Co.] 

By  a  saturated  solution  is  meant  one  in  which  the  liquid  has  dissolved  all 
that  it  can  of  the  substance  added.  This  varies  with  the  temperature.  It  is 
well  to  have  an  excess  of  the  substance  present  then  the  liquid  will  be  satu- 
rated at  all  temperatures  usually  found  in  the  laboratory. 

\  375.  Solutions  less  than  10  per  cent. — In  making  solutions  where  dry 
substance  is  added  to  a  liquid  if  the  percentage  is  not  over  10%,  the  custom 
is  to  take  too  cc.  of  the  liquid  and  add  to  it  the  number  of  grams  indicated  by 
the  per  cent.  That  is  for  a  5%  solution  one  would  take  ico  cc.  of  the  liquid 
and  5  grams  of  the  dry  substance.  This  does  not  make  a  strictly  5%  solution. 
For  that  one  should  take  95  cc.  of  liquid  and  5  grams  of  the  dry  substance  ;  or 
if  the  percentage  must  be  exact  then  one  should  weigh  out  95  grams  of  the 
liquid  and  add  5  grams  of  the  dry  substance. 


Fi<;s.  218-219.     Scales  for  weighing  chemicals.     (Cuts  loaned  by  the  Bausch 
&  Lomb  Optical  Company.) 

§  376.     Solutions  of  10  per  cent  and  more. — When  the  percentage  is  10%  or 


270  PREPARATION  OF  REAGENTS  [CH.IX 

over  it  is  better  to  weigh  out  the  number  of  grams  representing  the  percentage 
and  add  to  it  the  right  amount  of  liquid  in  cubic  centimeters.  For  example 
if  one  were  to  make  a  35%  aqueous  solution  of  caustic  potash  in  water  then 
one  would  add  35  grams  of  caustic  potash  to  65  cc.  of  water.  If  one  wished  to 
make  a  10%  alcoholic  solution  of  caustic  potash  he  would  add  10  grams  of 
caustic  potash  to  90  cc.  of  alcohol.  But  here  is  a  case  where  the  alcohol  being 
of  less  specific  gravity  than  water  the  mixture  would  not  weigh  100  grams  ; 
and  to  make  the  mixture  weigh.ioo  grams  giving  therefore  an  exact  percentage, 
one  should  take  90  grams  of  alcohol  and  add  to  it  10  grams  of  caustic  potash. 
In  practice  in  making  solutions  of  collodion  or  celloidin  one  usually  mixes 
alcohol  and  95%  or  absolute  alcohol  in  equal  volumes  and  then  for  a  10% 
solution  10  grams  of  the  dry  soluble  cotton  or  celloidin  are  added  to  90  cc.  of 
the  ether-alcohol  mixture.  But  ether  is  much  lighter  than  water  and  the  alcohol 
somewhat  lighter,  so  that  the  percentage  in  this  case  would  be  more  than  10% 
because  the  90  cc.  of  alcohol  and  ether  would  weigh  considerably  less  than  90 
grams. 

\  377.  Mixtures  of  Liquids  to  Obtain  a  desired  Percentage. — It  frequently 
happens  that  it  is  desired  to  obtain  a  lower  percentage  or  strength  of  a  liquid 
than  the  one  in  stock.  This  is  very  readily  done  according  to  the  general  for- 
mula: Divide  the  percentage  of  the  strong  solution  by  the  percentage  of  the 
desired  solution  and  the  quotient  will  give  the  number  of  times  too  strong  the 
solution  is.  To  obtain  the  right  strength  take  I  of  the  strong  solution,  and  of 
the  diluting  liquid  one  less  than  the  quotient  obtained  by  dividing  the  per- 
centage of  the  strong  solution  by  the  percentage  of  the  weak  solution,  thus ; 
Suppose  it  is  desired  to  obtain  a  5%  solution  of  formaldehyde.  As  the  strong 
solution  obtainable  in  the  market  is  a  40%  aqueous  solution  of  formaldehyde 
gas  it  is  8  times  too  strong  for  the  desired  solution.  To  get  the  proper 
strength  one  takes  i  cc.  of  the  40%  formaldehyde  and  adds  to  it  7  cc.  of  water 
and  the  resulting  mixture  will  be  only  *i  the  strength  of  the  original  solution 
or  5%  instead  of  40%. 

§  378.  Mixtures  of  Alcohol. — For  alcohol  if  one  desires  a  50%  solution  it 
is  usually  near  enough  correct  to  add  equal  parts  of  95%  alcohol  and  water,  but 
this  does  not  actually  give  a  50%  solution.  To  find  the  real  proportions 
according  to  the  general  formula  :  95%-=-5o%— 1.9  i.  e.y  for  every  i  cc.  of  95% 
alcohol  should  be  added  0.9  cc.  of  water  or  for  each  100  cc.  of  95%  alcohol,  90 
cc.  of  water.  This  even  will  not  give  an  exact  mixture  of  alcohol  for  a  mix- 
ture of  alcohol  and  water  diminishes  somewhat  in  volume.  To  get  true  per- 
centages an  alcoholometer  for  testing  the  specific  gravity  is  used. 

A  simple  method  of  getting  approximately  correct  mixtures  of  alcohol  is 
the  following :  Pour  the  strong  alcohol  into  a  graduate  glass  (Fig.  215-217) 
until  the  volume  is  the  same  as  the  desired  percentage,  then  add  water  until 
the  volume  is  the  same  as  the  original  percentage  of  the  alcohol.  Example  : 
To  get  50%  from  95%  alcohol  put  50  cc.  of  95%  into  a  graduate  and  fill  the 
graduate  to  95  cc.  with  water,  and  the  resulting  mixture  will  be  50%  alcohol, 
and  so  with  all  other  strengths.  Here  the  shrinkage  is  eliminated  from  con- 
sideration because  the  water  and  alcohol  are  not  measured  separately  and  then 
mixed,  but  one  is  added  to  the  other  until  a  given  volume  is  attained. 


Lll.   /.V]  PREPARATION  OF  REAGENTS  271 

SOME  OF  THE  MORE  IMPORTANT  REAGENTS  USED  IN  MICROSCOPY 

i;  379.  Albumen  Fixative  (Mayer's). — This  consists  of  equal  parts  of 
well-beaten  white  of  egg  and  glycerin.  To  each  50  cc.  of  this  i  gram  of  salicy- 
late  of  soda  is  added  to  prevent  putrefactive  changes.  This  must  be  carefully 
filtered.  For  method  of  use  see  Ch.  X.  $  448. 

\  380.  Alcohol  (Ethyl),  C,,  H-,  O  H. — Ethyl  or  grain  alcohol  is  mostly 
used  for  histologic  purposes.  (A)  absolute  alcohol  (i.  e. ,  alcohol  of  99%) 
is  recommended  for  many  purposes,  but  if  plenty  of  95%  alcohol  is  used  it 
answers  every  purpose  in  histology,  in  a  dry  climate  or  in  a  warm,  dry  room. 
When  it  is  damp,  dehydration  is  greatly  facilitated  by  the  use  of  absolute 
alcohol. 

(B)  82%  alcohol  made  by  mixing  5  parts  of  95%    alcohol   with    I    part   of 
water. 

(C)  67%  alcohol  made  by  mixing  2  parts  of  95%    alcohol   with    i    part   of 
water.     See  also  \  378-379. 


FIG.  220.     Reagent  bottle.      (Cut  loaned  by  the    Whitall 
Tat  urn  Co.) 


\  381.  Alcohol  (Methyl)  C-H.,,  O  H.— Methyl  alcohol  or  wood  alcohol 
is  much  cheaper  than  ethyl  or  grain  alcohol  on  account  of  the  revenue  tax  on 
ethyl  alcohol.  It  answers  well  for  many  microscopic  purposes.  It  has  been 
refined  so  carefully  in  recent  years  that  the  disagreeable  odor  is  not  very 
noticeable. 

'',.  3*2.  Denatured  Alcohol.— This  is  Ethyl  or  grain  alcohol  rendered  un- 
drinkable  by  the  addition  of  wood  alcohol  and  benzine  (Grain  alcohol,  S$}4%  ; 
Methyl  alcohol  10%,  and  Benzine  )4%)-  In  some  cases  the  denaturing  sub- 
stances are  somewhat  different,  but  all  render  the  alcohol  unusable  for  drink- 
ing. It  is  then  free  from  internal  revenue  tax. 

In  Great  Britain  "  Methylated  Spirits"  consists  of  grain  alcohol  with  10% 
methyl  alcohol.  This  is  used  very  largely  in  microscopic  work.  In  America 
the  addition  of  the  Ben/.ine  renders  denatured  alcohol  also  unfit  for  histologi- 
cal  purposes  if  it  is  to  be  diluted.  The  addition  of  water  makes  it  milky.  If 
methyl  alcohol  alone  or  combined  with  pyridin  or  some  other  substance  wholly 


272  PREPARA  TION  OF  REAGENTS  [  CH.  IX 

soluable  in  water  were  used  as  the  denaturing  substance,  denatured  alcohol 
could  be  used  in  microscopic  work  for  all  the  grades.  That  denatured  as  indi- 
cated above  can  be  used  only  in  full  strength  or  very  slightly  diluted. 

For  educational  and  other  public  institutions  the  U.  S.  government  grants 
the  privilege  of  using  ethyl  alcohol  without  paying  the  revenue  tax,  but  for 
private  institutions  and  for  individuals  it  would  be  a  great  relief  if  the  dena- 
tured alcohol  could  be  mixed  in  all  proportions  with  water  without  the  forma- 
tion of  precipitates. 

\  383.  Balsam,  Canada  Balsam,  Balsam  of  Fir. — This  is  one  of  the  oldest 
and  most  satisfactory  of  the  resinous  media  used  for  mounting  microscopic 
preparations. 

The  natural  balsam  is  most  often  used  ;  it  has  the  advantage  of  being  able 
to  take  up  a  small  amount  of  water  so  that  if  sections  are  not  quite  dehydrated 
they  will  clear  up  after  a  time. 

\  384.  Xylene  Balsam. — This  is  Canada  Balsam  diluted  or  thinned  with 
xylene  (xylol  of  the  Germans).  It  is  recommended  by  many  to  evaporate  the 
natural  balsam  to  dryness  and  then  to  dissolve  it  in  xylene.  For  some  pur- 
poses, e.  g.:  for  mounting  glycogen  preparations,  this  is  advantageous  ;  but  it 
is  unnecessary  for  most  purposes.  Xylene  balsam  requires  a  very  complete 
desiccation  or  dehydration  of  objects  to  be  mounted  in  it  for  the  xylene  is 
immiscible  with  water. 

\  385.  Filtering  Balsam.  Balsam  is  now  furnished  already  filtered 
through  filter  paper.  If  xylene  balsam  is  used  it  may  be  made  thin  and 
filtered  without  heat.  For  filtering  balsam  and  all  resinous  and  gummy 
materials,  the  writer  has  found  a  paper  funnel  the  most  satisfactory.  It  can 
be  used  once  and  then  thrown  away.  Such  a  funnel  may  be  easily  made  by 
rolling  a  sheet  of  thick  writing  paper  in  the  form  of  a  cone  and  cementing  the 
paper  where  it  overlaps,  or  winding  a  string  several  times  around  the  lower 
part.  Such  a  funnel  is  best  used  in  one  of  the  rings  for  holding  funnels,  so 
common  in  chemical  laboratories.  The  filtering  is  most  successfully  done  in 
a  very  warm  place  like  an  incubator  or  an  incubator  room. 

\  386.  Neutral  Balsam. — All  the  samples  of  balsam  tested  by  the  author 
have  been  found  slightly  acid.  This  is  an  advantage  for  carmine,  and  acid 
fuchsin  stain  or  any  other  acid  stain.  Also  for  preparations  injected  with 
carmine  or  Berlin  blue.  In  these  cases  the  color  would  fade  or  diffuse  if  the 
medium  were  not  slightly  acid.  For  hematoxylin  and  many  other  stains  the 
acid  is  detrimental.  For  example,  the  slight  amount  of  acid  in  the  balsam 
causes  the  delicate  stain  in  the  finest  fibers  of  Weigert  preparations  to  fade. 
To  neutralize  the  balsam  add  some  pure  sodium  carbonate,  set  the  balsam  in  a 
warm  place  and  shake  it  occasionally.  After  a  month  or  so  the  soda  will 
settle  and  the  clear  supernatant  balsam  will  be  found  very  slightly  alkaline. 
Use  this  whenever  an  acid  medium  would  fade  the  stain  in  the  specimen. 

\  387.  Acid  Balsam. — As  stated  above  all  balsam  is  naturally  somewhat 
acid,  but  for  various  stains  it  is  desirable  to  increase  the  acidity.  For 
example,  specimens  stained  with  picro-fuchsin,  or  injected  with  carmine  or 


CH.  IX}  PREPARATION  OF  REACENTS  273 

Berlin  blue  are  more  satisfactory  and  last  longer  with  full  brilliancy  if  the 
balsam  is  made  more  acid  than  it  naturally  is.  For  this  use  10  to  20  drops  of 
glacial  acetic  or  formic  acid  to  100  cc.  of  balsam. 

\  388.  Borax  Carmine  for  in  Toto  Staining. — Borax  4  grams  ;  Carmine  3 
grams ;  water  100  cc.  Shake  frequently  for  several  days  and  then  filter  and 
add  100  cc.  of  67%  alcohol.  After  3  to  4  days  it  may  be  necessary  to  filter 
again.  Good  for  in  toto  staining  after  almost  any  fixer.  Put  the  object  to  be 
stained  from  alcohol  into  a  vial  with  plenty  of  stain.  After  a  day  or  two 
change  the  stain.  Stain  4  to  5  days.  Remove  to  67%  alcohol  adding  4  drops 
of  H  Cl  to  each  100  cc.  of  alcohol.  After  one  day  remove  to  82%  alcohol. 
Change  the  alcohol  till  no  more  color  comes  away,  then  proceed  to  section. 
Remember  that  objects  stained  in  toto  may  be  mounted  directly  in  balsam 
from  de-paraffining  xylene. 

$  389.  Carmine  for  Mucus  (Mucicarmin). — One  can  buy  the  dry  powder 
or  preferably  prepare  the  stain.  To  prepare  it  take  i  gram  of  Carmine  No.  40 
and  YZ  gram  of  pure  dry  ammonium  chlorid.  If  the  latter  is  slightly  moist, 
dry  it  in  an  evaporating  dish  in  a  sand  bath.  Mix  the  ammonium  chlorid  and 
the  carmine  and  add  2  cc.  of  water.  Mix  well  and  heat  over  a  sand  bath,  con- 
stantly mixing  with  a  glass  rod.  Continue  the  heating  until  the  carmin  col- 
ored mass  becomes  very  dark  red.  It  will  take  3  to  10  minutes  for  this.  The 
heat  should  not  be  too  great. 

Dissolve  the  dark  red  mixture  in  100  cc.  of  50%  alcohol.  For  use,  dilute 
five  or  tenfold  with  tap  water.  This  stains  best  after  mercuric  fixers.  One 
must  not  collodionize  sections  to  be  stained  with  this  as  the  carmine  stains  the 
collodion  very  deeply.  Stain  the  sections  first  with  hematoxylin  as  usual 
then  stain  i  to  5  hours  or  longer  with  the  dilute  mucicarmin.  The  mucus  in 
goblet  cells,  in  the  mucous  part  of  the  salivary  glands,  etc.,  will  be  red. 
Nuclei  will  be  stained  with  hematoxylin.  Mount  in  balsam  (\  383). 

\  390.  Cedar- Wood  Oil. — This  is  used  for  oil  immersion  objectives  and  is 
quite  thick. 

For  penetrating  tissues  and  preparing  them  for  infiltration  with  paraffin, 
thick  oil  is  recommended  by  Lee.  The  writer  has  found,  however,  that  any 
good  cedar-wood  oil  gives  excellent  results  in  ordinary  histologic  and  embry- 
ologic  work.  That  known  as  Cedar- Wood  Oil  (Florida)  is  excellent,  also 
that  known  as  Cedar- Wood  Oil  (true  Lebanon).  These  forms  are  far  less 
expensive  than  that  used  for  immersion  objectives.  The  tissues  should  be 
thoroughly  dehydrated  before  putting  them  into  cedar-wood  oil,  and  they 
should  remain  until  they  are  translucent. 

?.  391.  Clarifier,  Castor-Xylene  Clarifier. — This  is  composed  of  castor  oil  i 
part  and  xylene*  3  parts.  (Trans.  Amer.  Micr.  Soc.,  1895,  p.  361.) 


*The  hydrocarbon,  xylene  (CMH10)  is  called xylol  in  German.  In  English, 
members  of  the  hydrocarbon  series  have  the  termination  "  ene  "  while  mem- 
bers of  the  alcohol  series  terminate  in  "  ol." 


274  PREPARA  TION  OF  REAGENTS  [  CH.  IX 

\  392.  Carbol-Xylene  Clearer. — Vasale  recommends  as  a  clearer,  xylene 
75  cc. ,  carbolic  acid  (melted  crystals)  25  cc. 

\  393.  Carbol-Turpentine  Clearer. — A  satisfactory  and  generally  applica- 
ble clearer  is  carbol-turpentine,  made  by  mixing  carbolic  acid  crystals  (Aci- 
duni  carbolicum.  A.  phenicum  crystallizatum}  40  cc.  with  rectified  oil  of  tur- 
pentine (Oleum  terebinthinae  rectificatutri)  60  cc.  If  the  carbolic  acid  does 
not  dissolve  in  the  turpentine,  increase  the  turpentine,  thus  :  carbolic  acid  30 
cc.,  turpentine  70  cc. 

This  clearer  is  not  so  good  as  the  preceding  for  mounting  objects  which 
have  been  stained  with  osmic  acid  as  the  hydrogen  dioxid  (H.,O.,)  present 
fades  the  blackened  osmic  acid. 

'',.  394.  Collodion. — This  is  a  solution  of  soluble  cotton*  or  other  form  of 
pyroxylin  in  equal  parts  of  sulfuric  ether  and  95%  or  absolute  alcohol. 
Four  solutions  are  used  for  infiltrating  and  imbedding. 

(1)  i>2%  Collodion.     95%   or  absolute  alcohol   100  cc.;  soluble    cotton 
3  grams.     Let  the  cotton  soak  well  in  the  alcohol  and  then  add  100  cc.    of   sul- 
furic ether. 

(2)  2>%    Collodion.     Soluble  cotton  3  grams.     95%   or  absolute   alcohol 
50  cc.     After  the  cotton  has  become  well  wet  with  the  alcohol   add  50   cc.    of 
sulfuric  ether. 

(3)  6%  Collodion.     For  this  take  6  grams  of  soluble  cotton  and  50  cc.  of 
absolute  alcohol.     Let  the  cotton  remain  in  the  alcohol  over  night  and   then 
add  the  50  cc.  of  sulfuric  ether. 

(4)  8%  Collodion.     Take  8  grams  of   soluble  cotton  and  50  cc.  of  absolute 
alcohol.     Leave  the  cotton  in  the  alcohol  over  night  or  longer   and   then   add 
50  cc.  of  sulfuric  ether. 


*The  substance  used  in  preparing  collodion  goes  by  various  names,  soluble 
cotton  or  collodion  cotton  is  perhaps  best.  This  is  cellulose  nitrate,  and  consists 
of  a  mixture  of  cellulose  tetranitrate  C]2H](;(NO.,)4O6,  and  cellulose  pentani- 
trate,  Ci;;Hj-(NOs):)C>5.  Besides  the  names  soluble  and  collodion  cotton,  it  is 
called  gun  cotton  and  pyroxylin.  Pyroxylin  is  the  more  general  term  and  in- 
cludes several  of  the  cellulose  nitrates.  Celloidin  is  a  patent  preparation  of 
pyroxylin,  more  expensive  than  soluble  cotton. 

Soluble  cotton  should  be  kept  in  the  dark  to  avoid  decomposition.  After 
it  is  in  solution  this  decomposition  is  not  so  liable  to  occur.  The  decomposi- 
tion of  the  dry  cotton  gives  rise  to  nitrous  acid,  and  hence  it  is  best  to  keep  it 
in  a  box  loosely  covered  so  that  the  nitrous  acid  may  escape. 

Cellulose  nitrate  is  explosive  under  concussion  and  when  heated  to  150° 
centigrade.  In  the  air,  the  loose  soluble  cotton  burns  without  explosion.  It 
is  said  not  to  injure  the  hand  if  held  upon  it  during  ignition  and  that  it  does 
not  fire  gun  powder  if  burned  upon  it.  So  far  as  known  to  the  writer,  no  acci- 
dent has  ever  occurred  from  the  use  of  soluble  cotton  for  microscopic  pur- 
poses. I  wish  to  express  my  thanks  to  Professor  W.  R.  Orndorff,  organic 
chemist  in  Cornell  University,  for  the  above  information.  Proc.  Amer.  Micr. 
Soc.,  vol.  XVII  (1895),  pp.  361-370. 


CH.  AY]  PREPARATION  OF  REACENTS  275 

.\\\  collodion  solutions  should  be  kept  well  corked  or  the  ether  will  evapo- 
rate, also  some  of  the  alcohol,  and  leave  the  soluble  cotton  as  a  kind  of  jelly. 

j;  395.  Collodion  for  Cementing  Sections  to  the  Slide. — This  is  a  %%  so- 
lution made  by  adding  %  gram  of  soluble  cotton  to  50  cc.  of  95%  or  absolute 
alcohol  and  50  cc.  of  sulfuric  ether.  This  may  be  used  for  spreading  on  the 
sections  before  deparaffining  or  preferably  afterward. .  See  $  450. 

'',.  396.  Congo  Red. — Water  100  cc. ,  Congo  red  }^  gram.  This  is  a  good 
counter  stain  for  hematoxylin. 

\  397.  Congo-Glycerin. — For  mixing  with  and  staining  isolation  prepara- 
tions ($  357-361)  and  for  a  mounting  medium  this  is  an  excellent  combina- 
tion. It  is  particularly  good  for  nerve  cells. 

\  398.  Decalcifier. — For  removing  the  salts  of  lime  from  bone  etc.  One 
must  first  fix  and  harden  the  tissue  by  some  approved  method.  67%  Alcohol  loo 
cc.;  strong  nitric  acid  3  cc.  Change  two  or  three  times.  It  takes  from  3  to  10 
days  depending  on  the  object.  One  can  tell  when  the  decalcification  is  com- 
plete by  inserting  a  needle.  If  there  is  no  gritty  feeling  the  work  is  done. 
Then  wash  a  few  minutes  in  water  and  transfer  to  67%  alcohol.  Then  after  24 
hours  use  82%  alcohol.  It  is  usually  better  to  section  by  the  collodion  method. 
Tissue  is  liable  to  deteriorate  after  being  decalcified,  so  section  it  soon. 

I  399.  Dissociating  Liquids. — These  liquids  are  for  preserving  the  tissue 
elements  or  cells  and  for  dissolving  or  softening  the  intercellular  substance  so 
that  the  cells  may  be  readily  separated  from  their  neighbors.  The  separation 
is  accomplished  by  (a)  teasing  with  needles  ;  (b)  shaking  in  a  liquid  in  a  test 
tube  ;  (c)  scraping  with  a  scalpel  and  crushing  with  the  flat  of  the  blade  ;  (d) 
by  tapping  sharply  on  the  cover-glass  after  the  object  is  mounted.  One  may 
find  it  desirable  to  use  (d)  with  all  the  methods. 

(1 )  Formaldehyde  Dissociator. — Strong  formalin  (40%  formaldehyde  gas 
in  water)  2  cc.     Normal  salt  solution  1000  cc.     One  can  begin  work   within  ^ 
hour  and  good  results  may  be  obtained  after  2  to  3  days  immersion.     Excellent 
for  epithelia  and  for  nerve  cells. 

(2)  Miiller's  Fluid  Dissociator. — Miiller's  Fluid  i  cc.     Normal  salt  solu- 
tion 9  cc.    It  usually  requires  from  i  to  5  days  for  epithelia  to  dissociate  in  this. 
The  action  is  more  rapid  in  a  warm  place. 

(3)  Nitric  Acid  Dissociator.— Nitric  Acid  20  cc.     Water   So  cc.     This   is 
nsed  especially  for  muscular  tissue.     It  takes  from  one  to   3  days  depending 
on  the  temperature.     The  nitric  acid  gelatinizes  the  connective  tissue.     Wash 
out  the  acid  with  water.     Preserve  in  2  %  formaldehyde. 

\  400.  Elastic  Stain. — For  staining  elastic  substance  the  Resorcin  basic- 
fuchsin-Iron-Chlorid  of  Weigert  is  available.  The  stain  is  prepared  as  fol- 
lows. 

Basic  Fuchsin  2  grams.  Resorcin  4  grams.  Water  200  cc.  Boil  for 
several  minutes  (5  to  10).  Add  to  the  boiling  mixture  25  cc.  of  a  30%  aqueous 
solution  of  chlorid  of  iron  (Fe  Cl  6).  Boil  for  3  to  10  minutes  then  add  a 
saturated  solution  of  iron  chlorid  until  the  color  is  all  precipitated.  Try  the 


276  PREPARATION  OF  REAGENTS  [  CII.  IX 

liquid  occasionally  by  letting  a  few  drops  run  down  the  side  of  the  glass 
beaker  used  for  the  boiling.  If  the  color  is  precipitated  it  appears  as  fine 
granules  and  the  liquid  is  almost  uncolored  or  slightly  yellow. 

Allow  the  liquid  to  cool.  If  there  is  plenty  of  time  let  it  stand  over  night. 
Then  either  pour  off  the  supernatant  liquid  or  if  the  precipitate  has  not  set- 
tled filter  through  filter  paper.  Then  either  scrape  off  the  precipitate  from 
the  filter  paper  or  cut  off  the  lower  end  of  the  filter  containing  the  precipitate 
and  put  it  in  the  beaker.  Add  200  cc.  of  95%  alcohol  and  heat  over  a  water  bath 
till  the  alcohol  boils.  Continue  the  boiling  5  minutes  or  more  and  stir  up  the 
filter  paper  so  that  all  the  precipitate  may  be  dissolved.  After  boiling  5  minutes 
or  more  filter  the  hot  alcoholic  solution  into  a  warmed  bottle.  After  this 
alcoholic  solution  is  cool  add  5  cc.  of  strong  hydrochloric  acid. 

Stain  sections  in  this  solution  i  hour  sometimes  less.  Wash  off  the  stain 
with  95%  alcohol. 

This  works  well  on  sections  by  the  paraffin  or  the  collodion  method  and 
for  tissues  hardened  in  any  manner. 

§  401.  Eosin. — This  is  used  mostly  as  a  contrast  stain  with  hematoxylin, 
which  is  an  almost  purely  nuclear  stain.  It  serves  to  stain  the  cell-body, 
ground  substance,  etc.,  which  would  be  too  transparent  and  invisible  with 
hematoxylin  alone.  If  eosin  is  used  alone  it  gives  a  decided  color  to  the  tis- 
sue and  thus  aids  in  its  study.  Eosin  is  used  in  alcoholic  and  in  aqueous  solu- 
tions. A  very  satisfactory  stain  is  made  as  follows  :  50  cc.  of  water  and  50  cc. 
of  95%  alcohol  are  mixed  and  i-io  of  a  gram  of  dry  eosin  added.  V',,  aque- 
ous eosin  is  also  good. 

\  402.  Eosin  in  95  per  cent  Alcohol. — For  staining  embryos  and  tissues 
so  that  the  tissue  in  the  ribbons  of  sections  may  be  easily  seen  a  saturated 
solution  of  alcoholic  eosin  is  made.  This  is  also  used  for  staining  with 
methylene  blue  (see  \  471). 

I  403.  Ether,  Ether-Alcohol. — Sulfuric  ether  is  meant  when  ether  is 
mentioned  in  this  book.  Wherever  ether-alcohol  is  mentioned  it  means  a 
mixture  of  equal  volumes  of  sulfuric  ether  and  95%  or  absolute  alcohol. 

§  404.  Farrant's  Solution. — Take  25  grams  of  clean,  dry,  gum  arabic, 
25  cc.  of  a  saturated  aqueous  solution  of  arsenious  acid  ;  25  cc.  of  glycerin. 
The  gum  arabic  is  soaked  for  several  days  in  the  arsenic  water,  then  the 
glycerin  is  added  and  carefully  mixed  with  the  dissolved  or  softened  gum 
arabic. 

This  medium  retains  air  bubbles  with  great  tenacity.  It  is  much  easier  to 
avoid  than  to  get  rid  of  them  in  mounting. 

\  405.  Flemming's  Fluid. — Water  19  cc. ;  i%'  osmic  acid  10  cc. ;  io% 
chromic  acid  3  cc  ;  Glacial  acetic  acid  2  cc.  This  osmic  fixer  is  good  for  very 
small  pieces,  i  to  5  millimeter  pieces  ;  thickness  not  over  2  to  3  mm.  Wash 
out  with  water  10  to  24  hours.  Then  67%  alcohol.  Also  82%  and  95%. 

§  406.  Formaldehyde  (H.  CHO  or  OCH,.) — This  is  found  in  the  market 
under  the  name  of  "formalin,"  etc.,  and  consists  of  a  40%  solution  of  for- 
maldehyde gas  in  water. 


C/I.  /A']  PREPARATION  OF  KE  AGENTS  277 

For  fixing  tissues  and  embryos  a  5%  solution  is  good  (Formalin  i  cc. , 
water  7  cc.,  \  377).  A  common  fixer  is  10  cc.  formalin,  90  cc.  water.  This  is 
frequently  called  10",,  formalin,  it  is  however  only  4%  formaldehyde. 

Tissues  may  stay  in  this  indefinitely.  Small  pieces  are  fixed  within  an 
hour.  Before  hardening  in  alcohol  and  imbedding,  wash  out  the  formalin  in 
running  water  half  an  hour,  then  harden  a  day  or  more  in  67%  and  82% 
alcohol. 

For  preserving  nitric  acid  dissociated  muscle  a  2%  formaldehyde  solution 
is  i;ood.  (Formalin  I  cc. ,  water  19  cc.  \  377.)  See  also  \  399  (i)  for  the  for- 
maldehyde dissociator. 

''/.  407.  Glycerin. — (A.)  One  should  have  pure  glycerin  for  a  mounting 
medium.  It  needs  no  preparation,  unless  it  contains  dust  when  it  should  be 
filtered  through  filter  paper  or  absorbent  cotton. 

To  prepare  objects  for  final  mounting,  glycerin  50  cc.,  water  50  cc.,  forms 
a  good  mixture.  For  many  purposes  the  final  mounting  in  glycerin  is  made 
in  an  acid  medium,  vi/.,  Glycerin  99  cc. ,  Glacial  acetic  or  formic  acid,  i  cc. 

By  extreme  care  in  mounting  and  by  occasionally  adding  a  fresh  coat  to 
the  sealing  of  the  cover-glass,  glycerin  preparations  last  a  long  time.  They 
are  liable  to  be  disappointing,  however.  In  mounting  in  glycerin  care  should 
be  taken  to  avoid  air-bubbles,  as  they  are  difficult  to  get  rid  of.  A  specimen 
need  not  be  discarded,  however,  unless  the  air-bubbles  are  large  and  numerous. 
See  also  Congo  glycerin  \  397. 

\  408.  Glycerin  Jelly  for  Microscopic  Specimens. — Soak  25  grams  of  the 
best  dry  gelatin  in  cold  water  in  a  small  agate-ware  dish.  Allow  the  water  to 
remain  until  the  gelatin  is  softened.  It  usually  takes  about  half  an  hour. 
When  softened,  as  may  be  readily  determined  by  taking  a  little  in  the  fingers, 
pour  off  the  superfluous  water  and  drain  well  to  get  rid  of  all  the  water  that 
has  not  been  imbibed  by  the  gelatin.  Warm  the  softened  gelatin  over  a  water 
bath  and  it  will  melt  in  the  water  it  has  absorbed.  Add  about  5  cc.  of  egg 
albumen,  white  of  egg  ;  stir  it  well  and  then  heat  the  gelatin  in  the  water  bath 
for  about  half  an  hour.  Do  not  heat  above  75°  or  80°  C.,  for  if  the  gelatin  is 
heated  too  hot  it  will  be  transformed  into  meta-gelatin  and  will  not  set  when 
cold.  Heat  coagulates  the  albumen  and  it  forms  a  kind  of  floculent  precipitate 
which  seems  to  gather  all  fine  particles  of  dust,  etc.,  leaving  the  gelatin  per- 
fectly clear.  After  the  gelatin  is  clarified,  filter  through  a  hot  flannel  filter 
and  mix  with  an  equal  volume  of  glycerin  and  5  grams  of  chloral  hydrate  and 
shake  thoroughly.  If  it  is  allowed  to  remain  in  a  warm  place  (i.  e.,  in  a  place 
where  the  gelatin  remains  melted)  the  air-bubbles  will  rise  and  disappear. 

In  case  the  glycerin  jelly  remains  fluid  or  semi-fluid  at  the  ordinary  tem- 
perature ( i8°-2o°  C.),  the  gelatin  has  either  been  transformed  into  meta-gela- 
tin by  too  high  a  temperature  or  it  contains  too  much  water.  The  amount  of 
water  may  be  lessened  by  heating  at  a  mbderate  temperature  over  a  water  bath 
in  an  open  vessel.  This  is  an  excellent  mounting  medium.  Air-bubbles 
should  be  avoided  in  mounting  as  they  do  not  disappear. 

\  409.  Glycerin  Jelly  for  Anatomic  Preparations.— Specimens  prepared 
by  the  Kaiserling  method  or  other  satisfactory  way  may  be  permanently  pre- 


278  PREPARATION  OF  REAGENTS  \_CH.IX 

served  in  glycerin  jelly  prepared  as  follows  :  Best  clear  gelatin,  200  grains. 
Kaiserling's  No.  4  solution,  3000  cc.  (Potassium  acetate,  100  grams  ;  glycerin, 
200  cc.;  water,  1000  cc.)  Put  the  gelatin  in  the  potassium-acetate-glycerin- 
water,  mixture  in  an  agate  pail  and  heat  over  a  gas  or  other  stove.  Stir.  When 
the  temperature  is  about  55°  centigrade  add  the  whites  of  three  eggs  well 
beaten,  and  stir  them  in  vigorously.  Make  markedly  acid  by  acetic  acid. 
Continue  the  heating  until  the  mixture  just  boils,  and  then  filter  through  filter 
paper  into  fruit  jars.  It  is  best  to  put  over  the  filter  paper  two  thicknesses  of 
gauze  (g  330).  A  piece  of  thymol  in  the  top  of  each  jar  will  prevent  the 
growth  of  fungi,  or  one  can  add  5%  chloral  hydrate.  Specimens  are  mounted 
in  this  jelly  directly  from  the  No.  4  Kaiserlings,  or  alcoholic  specimens  can 
be  soaked  in  water  an  hour  or  more  and  then  kept  in  some  of  the  melted  jelly 
until  well  soaked,  then  mount  permanently  in  the  glycerin  jelly.  At  the  time 
of  mounting  the  gelatin  is  liquified  over  a  water  bath,  and  for  every  20  cc.  of 
the  gelatin  used  one  drop  of  strong  formalin  is  added.  This  is  to  prevent  the 
liquifaction  of  the  gelatin  after  the  specimen  is  mounted.  Let  the  gelatin 
cool  gradually  after  the  specimen  is  in  place,  then  add  some  melted  gelatin  to 
make  the  vessel  over  full  and  slide  a  glass  cover  on  it.  This  excludes  all  air. 
The  cover  may  then  be  sealed  with  the  clear  gelatin  or  glue  used  for  gluing 
wood,  or  the  cement  used  in  mending  crockery.  Finally  one  can  seal  with 
rubber  cement  if  desired.  (See  W.  H.  Walters,  N.  Y.  Med.  Record,  Dec. 
22,  1906.) 

\  410.  Chloral  Hematoxylin. — Potash  alum  4  grams.  Distilled  water 
125  cc.;  Hematoxylin  crystals  ^  gram.  Boil  5  to  10  minutes  in  an  agate  dish. 
After  cooling,  add  3  grams  of  chloral  hydrate  and  put  into  a  bottle.  This  will 
stain  more  rapidly  after  a  week  or  two  if  the  bottle  is  left  uncorked.  It  takes 
from  I  to  5  minutes  to  stain  sections.  Sometimes  a  longtime.  Use  after  any 
method  of  fixation. 

It  may  be  prepared  for  work  at  once  by  the  addition  of  a  small  amount  of 
hydrogen  dioxid  (H,O2). 

If  the  stain  is  too  concentrated  it  may  be  diluted  with  freshly  distilled 
water  or  with  a  mixture  of  water,  alum  and  chloral.  If  the  stain  is  not  suffi- 
ciently concentrared,  more  hematoxylin  may  be  added.  Proc.  Amer.  Micr. 
Soc.,  1892,  pp.  125-127). 

$  411.  Hematein.  This  is  used  instead  of  hematoxylin,  as  it  is  believed 
to  give  more  satisfactory  results.  Prepare  as  follows  :  Put  a  5%  solution  of  pot- 
ash alum  in  distilled  water  and  boil  or  leave  in  a  steam  steralizer  an  hour  or  two. 
While  warm  add  i  per  cent  of  hematein  dissolved  in  a  small  quantity  ot 
alcohol.  After  the  fluid  has  cooled  add  2  grams  of  chloral  for  each  100  cc.  of 
solution.  (Freeborn,  Jour.  Ap.  Micr.,  1900,  p.  1056.) 

$412.-  lodin  Stain  for  Glycogen. — lodin  i}4  gram  ;  iodid  of  potassium  3 
grams;  sodium  chlorid  \l/2  grams;  water  300  cc.  For  very  soluble  glycogen 
one  can  use  50%  alcohol  300  cc.  instead  of  water.  The  iodin  stain  is  the 
most  precise  and  differential  for  glycogen.  For  sectioning  tissues  or  embryos 
are  fixed  and  hardened  in  95%  or  absolute  alcohol.  Sectioned  by  the  paraffin 
method,  or  by  the  collodion  method,  but  for  permanent  preparations  the 


Cfl.  IX]  PREPARATION  OF  REAGENTS  279 

paraffin  method  is  best  (see  Ch.  X).  In  spreading  the  sections  use  this  iodin 
stain  instead  of  water.  Glycogen  in  the  sections  stains  a  mahogany  red,  and 
the  stain  remains  for  two  or  more  years  in  the  spread  paraffin  sections. 
Spread  sections  may  be  stained  or  restained  by  immersing  the  slide  in 
iodin  stain. 

Before  mounting  permanently  deparaffin  with  xylene,  and  mount  in 
melted  yellow  vaseline.  Press  the  cover  down  gently.  Seal  with  shellac  or 
balsam.  (Gage,  Trans.  Amer.  Micr.  Soc.,  1906.) 

\  413.  Iodin  in  Alcohol. — Iodin  10  grams  ;  95%  alcohol  90  cc.  This  is 
the  strong,  stock  solution. 

For  removing  the  pin-like  or  granular  mercuric  crystals  from  sections  of 
objects  fixed  in  any  fixer  containing  mercury  e.  g.,  Zenker's  fluid,  etc., 
take  95%  alcohol  500  cc.  and  the  10%  iodin  solution  5  cc.  In  some  cases 
where  the  amount  of  mercury  in  the  tissue  is  great  one  may  use  10  or  even 
15  cc.  of  the  strong  stock  solution.  Rinse  the  slide  well  in  pure  95%  alcohol 
to  remove  the  iodin  after  all  the  crystals  have  dissolved  (^  an  hour  or  more). 

For  embryos  and  tissues  fixed  in  a  mercuric  fixer  one  can  add  several 
drops  of  the  stock  solution  to  the  alcohol  containing  the  tissue  and  then  by 
changing  the  alcohol  occasionally  the  mercury  will  be  mostly  removed  before 
sectioning.  It  is  readily  removed  from  the  sections  as  just  described. 

§  4 [4.  Lamp-Black  for  Ingestion  by  Leucocytes. — Lamp-black,  2  grams  ; 
sodium  chlorid,  i  gram  ;  gum  acacia  (gum  Arabic),  i  gram  ;  distilled  water, 
100  cc.  Mix  all  thoroughly  in  a  mortar.  The  gum  arabic  is  to  aid  in  getting 
an  emulsion  of  the  lamp-black.  Filter  through  one  thickness  of  gauze  and 
one  of  lens  paper.  If  for  a  mammal  sterilize  by  boiling.  If  some  of  this 
mixture  is  injected  into  an  animal,  the  leucocytes  will  ingest  the  carbon  par- 
ticles. Carmine  may  be  used  instead  of  lamp-black,  but  it  is  not  as  good 
because  not  so  enduring  as  lamp-black. 

§  415.  Liquid  Gelatin. — Gelatin  or  clear  glue,  75  to  100  grams.  Com- 
mercial acetic  acid  (No.  8)  100  cc.,  water  100  cc.,  or  glacial  acetic  acid  40  cc. 
and  water  160  cc.,  95%  alcohol  100  cc. ,  glycerin  15  to  30  cc.  Crush  the  glue 
and  put  it  into  a  bottle  with  the  acid,  set  in  a  warm  place  and  shake  occasion- 
ally. After  three  or  more  days  add  the  other  ingredients.  This  solution  is 
excellent  for  fastening  paper  to  glass,  wood  or  paper.  The  brush  must  be 
mounted  in  a  quill  or  wooden  handle.  For  labels,  it  is  best  to  use  linen  paper 
of  moderate  thickness.  This  should  be  coated  with  liquid  gelatin  and  allowed 
to  dry.  The  labels  may  be  cut  of  any  desired  size  and  attached  by  simply 
moistening  them,  as  in  using  postage  stamps. 

Very  excellent  blank  labels  are  now  furnished  by  dealers  in  microscopic 
supplies,  so  that  it  is  unnecessary  to  prepare  them  one's  self,  except  for  special 
purposes.  Those  like  that  shown  in  Fig.  209  may  be  had  for  about  $3  for 
10,000. 

§  416.  Mercuric  Chlorid  (HgCl.,). — Mercuric  chlorid  7J<  grams;  sodium 
chlorid  i  gram  ;  water  too  cc.  The  solution  is  facilitated  by  heating  in  an 
agate  dish.  Fix  fresh  tissue  in  this  2  to  24  hours.  Then  transfer  to  67% 
alcohol  a  day  or  more  and  then  to  82%  alcohol.  Tissues  fixed  in  mercuric 


2So  PREPARATION  OF  REAGENTS  [  CH.  IX 

chlorid  deteriorate,  hence  it  is  better  to  imbed  them  soon  after  they  are  fixed. 
Crystals  of  mercury  are  removed  from  the  sections  by  the  use  of  iodized 
alcohol  (I  413). 

£  417.  Alkaline  Methylene  Blue. — Methylene  blue  2  grams  ;  95%  or  abso- 
lute alcohol  50  cc. ;  distilled  water  450  cc.;  \%  aqueous  caustic  potash  5  cc. 
This  stain  works  best  after  a  mercuric  fixer  or  a  fixer  containing  mercuric 
chlorid,  like  Zenker's  fluid. 

\  418.  Muller's  Fluid. — Potassium  dichromate  2^  grams;  sodium  sul- 
phate, i  gram  ;  water  100  cc.  This  is  one  of  the  oldest  fixers.  It  must  act  a 
long  time,  two  weeks  to  10  or  12  weeks.  This  longer  time  is  for  nervous  tissue 
to  be  stained  for  the  myelin.  Lately  this  fixer  has  been  combined  with  mer- 
cury (see  Zenker's  fluid  below).  Before  putting  the  tissue  into  67%  alcohol  it 
is  washed  out  in  running  water  for  24  hours. 

Muller's  Fluid  10  cc  ;  normal  salt  solution  90  cc.,  forms  an  excellent  disso- 
ciator  for  epithelia,  etc.  (§  399). 

\  419.  Neutral  Red.— This  is  used  especially  for  staining  living  animals. 
It  is  used  in  very  weak  solutions  :  -fa  gram  red  ;  1000  cc.  of  water.  Put  a  few 
cubic  centimeters  of  this  solution  into  the  vessel  containing  the  live  animal, 
or  animals.  Infusoria  stain  quickly  10  to  20  minutes  or  less.  Vertebrates 
may  require  a  few  days.  Try  it  on  infusoria  by  adding  a  drop  of  the  red  to 
several  drops  of  the  infusion  containing  the  infusoria.  Be  sure  that  there  are 
many  animals  present.  Watch  them  under  the  microscope  and  the  color  will 
be  seen  appearing  in  the  granules  of  the  infusoria.  Then  one  may  cover  and 
study  with  a  high  power. 

\  420.  Nitric  Acid,  H-NO... — This  is  employed  for  dissociation  (Nitric 
acid  Dissociator,  Water  80  cc.. ;  Nitric  acid  20  cc.);  as  a  fixer,  especially  for 
chick  embryos  in  the  early  stages  (Water  90  cc.;  Nitric  acid,  10  cc. ),  and  as  a 
decalcifier  (Nitric  acid  3  cc. ;  67%  alcohol  100  cc.). 

'',.  421.  Normal  Liquids.— A  normal  liquid  or  fluid  is  one  which  does  not 
injure  or  change  a  fresh  tissue  put  into  it.  The  perfect  normal  fluids  for  the 
tissues  of  any  animal  are  the  fluids  of  the  body  (lymph  and  plasma)  of  the 
animal  from  which  the  tissue  is  taken.  The  lymph  or  serum  of  one  species  of 
animal  may  be  far  from  normal  for  the  tissues  of  another  animal. 

The  commonly  used  artificial  normal  fluid  is  a  solution  of  common  salt 
(sodium  Chlorid)  in  water,  the  strength  varying  from  ^  to  ^  per  cent.  Aa 
indicated  above,  this  normal  salt  or  saline  solution  is  employed  in  diluting 
dissociating  liquids  (|  399). 

\  422.  Paraffin  Wax. — A  histologic  laboratory  requires  two  grades  of 
paraffin  for  ordinary  work.  These  are  hard  paraffin,  melting  at  about  54° 
centigrade,  and  a  softer  paraffin  melting  at  about  43°  centigrade.  Usually  a 
mixture  of  equal  parts  answers  very  well.  It  is  economical  for  a  laboratory  to 
buy  the  paraffin  wax  in  cases  of  about  200  pounds. 

All  paraffin  for  imbedding  and  sectioning  should  be  filtered  through  two 
thicknesses  of  filter  paper.  For  this,  use  a  metal  funnel,  heat  the  paraffin  very 


<  '//.  AV]  PREP.  IK.  I  TION  OF  REAGENTS  281 

hot  in  a  water  bath  and  then  heat  the  funnel  occasionally  with  a  Bunsen  flame. 
The  warmer  the  room  the  easier  to  filter  paraffin. 

Filter  the  paraffin  into  small  porcelain  pitchers.  If  the  paraffin  oven  has 
a  compartment  large  enough,  it  is  well  to  keep  one  of  the  pitchers  in  the  oven, 
then  the  paraffin  remains  melted  and  is  ready  for  use  at  any  time. 

\  423.  Picric-Alcohol. — This  is  an  excellent  hardener  and  fixer  foralmost 
all  tissues  and  organs.  It  is  composed  of  500  cc.  of  water  and  500  cc.  of  95% 
alcohol,  to  which  2  grams  of  picric  acid  have  been  added.  (It  is  a  -1%  solution 
of  picric  acid  in  50",,  alcohol).  It  acts  quickly,  in  from  one  to  three  daya. 
(Proc.  Ainer.  Micr.  Soc.,  Vol.  XII,  (1890),  pp.  120-122). 

\  424.  Picro-Fuchsin. — 10  cc.  of  a  i%  aqueous  solution  of  acid  fuchsin  ; 
75  cc.  of  a  saturated  aqueous  solution  of  picric  acid.  Stain  deeply  with  hema- 
toxylin  first,  then  use  the  picro-fuchsin.  Wash  off  the  picro-fuchsin  with  dis- 
tilled water.  Mount  in  non-neutralized  balsam  or  better  in  acid  balsam 
(Balsam  50  cc.  glacial  acetic  acid  5  drops).  If  the  white  connective  tissue  is 
not  red  enough  increase  the  amount  of  acid  fuchsin. 

;  425.  Shellac  Cement. — Shellac  cement  for  sealing  preparations  and  for 
making  shallow  cells*is  prepared  by  adding  scale  or  bleached  shellac  to  95% 
alcohol.  The  bottle  should  be  filled  about  half  full  of  dry  shellac  then  enough 
95",,  alcohol  added  to  fill  the  bottle  nearly  full.  The  bottle  is  shaken  occa- 
sionally and  then  allowed  to  stand  until  a  clear  stratum  of  liquid  appears  on 
the  top.  This  clear,  supernatant  liquid  is  then  filtered  through  filter  paper  or 
absorbent  cotton,  using  a  paper  funnel  ($  358),  into  an  open  dish  or  a  wide- 
mouth  bottle.  To  every  100  cc.  of  filtered  shellac  2  cc.  of  Venetian  turpen- 
tine may  be  added  to  render  it  less  brittle.  The  filtered  shellac  will  be  too 
thin,  and  must  be  allowed  to  evaporate  till  it  is  of  the  consistency  of  thin 
syrup.  It  is  then  put  into  a  capped  bottle,  and  for  use,  into  a  small  spirit 
lamp  (Fig.  203).  In  case  the  cement  gets  too  thick  add  a  small  amount  of 
95",,  alcohol  or  some  thin  shellac.  The  solution  of  shellac  almost  always  re- 
mains muddy,  and  in  most  cases  it  takes  a  long  time  for  the  flocculent  sub- 
stance to  settle.  One  can  quickly  obtain  a  clear  solution  as  follows  :  When 
the  shellac  has  had  time  to  thoroughly  dissolve,  /'.  e. ,  in  a  week  or  two  in  a 
warm  place,  or  in  less  time  if  the  bottle  is  frequently  shaken,  a  part  of  the  dis- 
solved shellac  is  poured  into  a  bottle  and  about  one-fourth  as  much  gasolin  or 
benzin  added  and  the  two  well  shaken.  After  twenty-four  hours  or  so  the 
flocculent,  undissolved  substance  will  separate  from  the  shellac  solution  and 
rise  with  the  gasolin  to  the  top.  The  clear  solution  may  then  be  siphoned  off 
or  drawn  off  from  the  bottom  if  one  has  an  aspirating  bottle.  (R.  Hitchcock, 
Amer.  Monthly  Micr.  Jour.,  July,  1884,  p.  131). 

If  one  desires  to  color  the  shellac,  the  addition  of  a  strong  alcoholic  solu- 
tion of  some  of  the  coal  tar  colors  is  good,  but  is  liable  to  dissolve  in  the 
mounting  medium  when  shellac  is  used  for  sealing,  A  small  amount  of  lamp- 
black well  rubbed  up  in  very  thin  shellac  and  filtered,  is  good  to  darken  the 
shellac. 

\  426.  Silvering. — Intercellular  substance  stains  brown  or  black  with 
nitrate  of  silver.  Use  '4  or  l/2%  aq.  sol.  on  fresh  tissue.  Stain  in  the  silver 


282  PREPARATION  OF  REAGENTS  [  C. H.  IX 

for  r  to  2  minutes  then  expose  to  light  in  water  till  brown.  One  may 
stain  afterward  with  hematoxylin  for  the  nuclei  ;  mount  in  glycerin,  glycerin 
jelly  or  in  balsam. 

\  427.  Sudan  III  for  Fat. — Sudan  III  or  azo-benzene-azo-/j-napthol,  was 
introduced  by  Daddi  into  histology  in  1896  (Arch.  Ital  de  Biologic,  t.  26.  p. 
142) ,  as  a  specific  stain  for  fat.  As  it  is  soluble  in  all  forms  of  fat  and  oils  and 
in  xylene,  alcohol,  etc.,  it  is  impossible  to  mount  specimens  in  balsam  after 
staining.  As  the  fat  of  tissues  is  removed  by  the  reagents  used  in  the  paraffin 
and  collodion  methods  (see  Ch.  X),  only  teased,  free-hand  or  frozen  sectioned 
material  fresh  or  fixed  in  some  non-fat  dissolving  fixer  can  be  used  (Miiller's 
fluid  and  5%  formaldehyde  are  excellent).  The  tissues  cut  free-hand  or  with 
the  freezing  microtome  or  teased  can  then  be  stained  with  a  saturated  alco- 
holic solution  of  the  Sudan.  It  stains  all  fat  a  brilliant  red.  Preparations  can 
be  preserved  in  glycerin  or  glycerin  jelly.  This  stain  is  largely  used  in 
Pathology. 

Daddi  used  the  substance  to  feed  animals  and  thus  to  stain  the  fat  which 
wyas  laid  down  in  the  body  while  the  Sudan  was  fed. 

The  fat  in  the  body  already  deposited  remains  unstained.  This  substance 
then  serves  to  record  the  deposit  of  fat  in  a  given  period.  In  1907  Dr.  Oscar 
Riddle  fed  Sudan  to  laying  hens,  and  the  fat  in  the  layers  of  yolk  laid  down 
during  the  feeding  was  stained  red  (Science,  XXVII,  1908,  p.  945).  For 
staining  the  yolks  of  hens  eggs  the  hen  may  be  fed  doses  of  20  to  25  milli- 
grams of  the  Sudan.  Eggs  so  colored  hatch  as  usual,  and  the  chick  in  utilix.- 
ing  the  colored  yolk  stains  its  body-fat  pink  (Susanna  P.  Gage). 

\  428.  Table  Black. — During  the  last  few  years  an  excellent  method  of 
dying  wood  with  anilin  black  has  been  devised.  This  black  is  lustreless,  and 
it  is  indestructible.  It  can  be  removed  only  by  scraping  off  the  wood  to  a 
point  deeper  than  the  stain  has  penetrated. 

It  must  be  applied  to  unwaxed  or  unvarnished  wood.  If  wax,  paint  or 
varnish  has  been  used  on  the  tables,  that  must  be  first  removed  by  the  use  of 
caustic  potash  or  soda  or  by  scraping  or  planing.  Two  solutions  are  needed  : 

SOLUTION   A 

Copper  sulphate 125  grams 

Potassium  chlorate  or  permanganate 125  grams 

Water looo'cc. 

,  Boil  these  ingredients  in  an  iron  kettle  until  they  are  dissolved.  Apply 
two  coats  of  the  hot  solution.  Let  the  first  coat  dry  before  applying  the 
second. 

SOLUTION    B 

Anilin  oil 120  cc. 

Hydrochloric' acid 180  cc. 

Water 1000  cc. 

Mix  these  in  a  glass  vessel  putting  in  the  water  first.  Apply  two  coats 
without  heating,  but  allow  the  first  coat  to  dry  before  adding  the  second. 


CH.  AY]  ri^l-.  PA  RATION  OI'' REAGENTS  283 

When  the  second  coat  is  dry,  sand  paper  the  wood  and  dust  off  the  excess 
chemicals.  Then  wash  the  wood  well  with  water.  When  dry,  sand  paper  the 
surface  and  then  rub  thoroughly  with  a  mixture  of  equal  parts  turpentine  and 
linseed  oil.  The  wood  may  appear  a  dirty  green  at  first  but  it  will  soon 
become  ebony  black.  If  the  excess  chemicals  are  not  removed  the  table  will 
crock.  An  occasional  rubbing  with  linseed  oil  and  turpentine  or  with  turpen- 
tine alone  will  clean  the  surface.  This  is  sometimes  called  the  Danish  method, 
Denmark  black  or  finish.  See  Jour.  Ap.  Micr.,  Vol.  I,  p.  145;  Bot.  Zeit.,  Vol. 
54,  p.  326,  Bot.  Gazette,  Vol.  24,  p.  66,  Dr.  P.  A.  Fish,  Jour.  Ap.  Micr.,  Vol. 

VI.,   pp.    2II-2I2. 

\  429.  Zenker's  Fluid. — Miiller's  Fluid,  (\  418),  loo  cc.;  mercuric 
chlorid  5  grams.  Just  before  using  add  5  cc.  of  glacial  acetic  acid  to  each 
100  cc.  of  the  above.  Fix  fresh  tissue  5  to  24  hours.  Wash  out  with  running 
water  24  hours.  Then  place  in  67%  alcohol  i  day  or  more  and  finally  preserve 
in  82%  alcohol.  Tissue  fixed  in  Zenker's  has  mercuric  crystals.  They  may 
be  removed  from  the  tissue  by  long  treatment  with  iodin,  or  by  putting  the 
slide  bearing  the  sections  in  iodized  alcohol  for  half  an  hour  or  more  (g  413). 

This  is  an  excellent  fixer,  combining  the  good  qualities  of  mercuric  chlorid 
and  of  the  chromium  compounds.  Tissues  fixed  with  this  show  well  the  red 
blood  corpuscles. 

REFERENCES    FOR   CHAPTER    IX 

For  information  concerning  this  chapter  the  reader  is  first  of  all  advised 
to  consult  the  microscopical  periodicals,  especially  the  Journal  of  the  Royal 
Microscopical  Society  and  the  Zeitschrift  fur  wissenschaftliche  Mikroskopie 
und  fiir  mikroskopische  Technik.  The  smaller  journals  and  the  proceedings 
of  microscopical  societies  frequently  have  excellent  articles  bearing  upon  the 
subjects  of  this  chapter.  This  is  especially  true  of  the  Journal  of  Applied 
Microscopy  and  Laboratory  Methods,  and  the  Transactions  of  the  American 
Microscopical  Society. 

Among  modern  books,  Lee's  Microtomists'  Vade  Mecum,  Mann's  Physio- 
logical Histology  and  Ehrlich's  Encyclopaedic  der  mikroskopischen  Technik 
are  indispensable  in  a  laboratory.  For  the  history  of  staining  see  Mann, 
pp.  190-195. 


CHAPTER  X 

FIXING  ;  MICROTOMES  AND  SECTION  KNIVES  ;  IMBED- 
DING ;  SECTIONING,  STAINING  AND  MOUNT- 
ING ;  SERIES  ;  MODELS 


FIXING   TISSUES,    ORGANS    AND    EMBRYOS  ;    MECHANICAL    PREPARA- 
TION   FOR    STUDY 

|  430.  Fixation. — By  fixing  or  fixation  in  histology  is  meant  the  prepara- 
tion of  fresh  tissues,  organs,  embryos  or  small  adult  animals  usually  by  means 
of  some  chemical  mixture,  called  a  "fixer"  so  that  the  organ  etc  as  a  whole 
and  the  elements  or  cells  composing  it  shall  retain  as  nearly  as  possible  the 
morphologic  characters  present  during  life.  The  more  perfect  the  fixer  the 
nearer  will  be  the  preservation  of  all  structural  details. 

Unfortunately  no  single  "fixer"  preserves  with  equal  excellence  all  the 
structural  details,  and  therefore  it  is  necessary  to  prepare  the  fresh  tissue  in 
several  different  ways  and  to  make  a  composite  of  the  structural  appearances 
found,  thereby  approximating  the  actual  structure  present  in  the  living  body. 
Changes  are  so  rapid  after  death  that  the  fixation  should  begin  as  soon  as  pos- 
sible. For  the  most  perfect  fixation  the  living  tissue  must  be  put  into  the 
fixer. 


FIGS.  221-222.  Class  stoppered  jars 
for  fixing  and  storing  tissues  for 
histology.  (Cuts  loaned  by  the  Whit- 
all  Tatum  Co.] 


With  one  of  the  larger  animals  where  the  whole  animal  is  to  be  used  for 
microscopic  study  it  is  a  great  advantage  to  bring  the  fixer  in  contact   with   all 


en.  X] 


FIXATION  OF  TISSl'ES 


285 


parts  of  the  body  quickly,  and  that  is  done  by  washing  out  the  vascular  sys- 
tem with  normal  salt  solution  and  then  filling  the  vascular  system  with  the 
fixer.  This  method  of  "  fixation  by  injection  "is  of  great  importance  in  the 
histology  of  animals  which  are  large  enough  to  inject. 

If  the  animal  is  too  small  for  injection  or  one  wishes  only  a  small  part  of 
a  larger  animal,  then  the  pieces  for  fixation  should  be  small,  say  one  to  three 
cubic  centimeters.  Often  as  for  Flemming's  fluid  (\  405)  and  for  several 
others  it  is  better  to  use  pieces  2  to  5  cubic  millimeters. 

Large,  solid  organs,  must  be  cut  into  several  pieces  if  the  whole  is  needed. 
For  hollow  organs  the  cavity  may  be  filled  with  the  fixer  and  the  organ  placed 
in  a  vessel  of  the  same. 


FIGS.  223-224.  Shell  vial  and  a  Comstock  bent-neck  vial  for  fixing  and 
storing  material  for  histology.  The  Comstock  bent-neck  vial  is  especially 
designed  for  elongated  objects  like  fish  embryos,  insects,  etc.,  which  are  liable 
to  become  bent  in  a  vertical  bottle.  (Cut  of  the  bent-neck,  from  the  Whitall 
Tatum  Co.) 

The  amount  of  fixer  should  be  10  to  50  times  that  of  the  piece  of  tissue. 

Of  the  fixers  given  under  "  Preparation  of  Reagents,"  Picric  alcohol, 
Formaldehyde  and  Zenker's  fluid  are  suitable  for  almost  every  tissue  and 
organ.  Formalin  has  the  advantage  of  having  strong  penetration,  hence  it 
preserves  whole  animals  fairly  by  immersing  after  filling  the  abdominal  and 
thoracic  cavities.  Formaldehyde  is  excellent  where  a  study  of  fat  is  in  ques- 
tion, and  it  is  much  used  as  a  fixer  where  frozen  sections  are  desired  (§  438). 
Remember  the  necessity  of  removing  mercury  from  sections  cf  tissues  fixed 
with  a  mercuric  fixer  (?  413,477). 

'{  431.  Mechanical  Preparation  of  Tissues  etc.  for  Microscopic  Study.  - 
A  limited  number  of  objects  in  nature  are  small  enough  and  transparent 


286 


MICROTOMES  AND  SECTION  KNIVES 


\_CH.  X 


enough,  and  a  limited  number  of  the  parts  of  higher  animals  are  suitable  for 
microscopic  study  without  mechanical  preparation  except  merely  mounting 
them  on  a  microscopic  slide.  Usually  the  parts  of  animals  are  so  large  and  so 
opaque  that  the  histologic  elements  or  cells  and  their  arrangement  in  organs 
can  only  be  satisfactorily  studied  with  a  microscope  after  the  tissue,  organ, 
etc.,  have  been  teased  apart  with  needles,  (£  357)  or  sectioned  into  thin  layers. 


FIG.  225 


FIG.  226 

FIGS.  225-226.  Washing  apparatus  for  tissues  fixed  in  osmic  and  chro- 
mium mixtures.  As  shown  in  the  figures  the  apparatus  is  connected  with  the 
zvater  pipe  by  a  small  side  cock.  It  is  composed  of  a  double  vessel,  the  inner 
one  being  made  of  perforated  brass.  There  are  special  perforated  dishes  to 
insert  in  the  little  compartments.  For  ova  and  other  small  objects  a  piece  of 
gauze  is  used  in  the  compartment.  This  apparatus  is  convenient  for  washing 
'cover-glasses,  for  the  washing  out  for  iron  hemato.vylin,  etc.  The  deeper  box 
at  the  right  answers  for  the  slide  baskets  or  holders  (Fig.  244). 

MICROTOMES    AND   SECTION    KNIVES 


\  432.  The  older  histologists,  those  who  laid  the  foundations  and  whose 
understanding  of  the  finer  structure  of  the  body  was  in  many  ways  superior  to 
the  knowledge  possessed  by  workers  at  the  present  time,  did  their  mechanical 


CH.  A']  MICROTOMES  AND  SECTION  KNIVES  287 

preparation  with  needles  and  with  sharp  knives  held  in  the  hand.  They  dealt 
also  with  fresh  tissue  more  largely  than  we  do  at  the  present  day,  and  learned 
also  to  distinguish  tissues  by  their  structure  rather  than  by  their  artificial 
coloration. 

It  was  not,  however,  on  account  of  the  lack  of  elaborate  mechanical  de- 
vices for  sectioning  and  complicated  staining  methods  of  the  present  day,  but 
because  they  put  intelligence  and  y.eal  into  their  work  that  made  them  so  suc- 
cessful. Only  those  who  were  "called"  made  for  themselves  a  laboratory  and 
saw  with  their  brain.  Now  many  are  "sent,"  but  few  who  use  the  central 
organ  of  sight. 

If  the  reader  is  interested  in  the  mechanical  means  for  sectioning  he  is 
referred  to  Dr.  C.  S.  Minot's  papers  on  the  history  of  the  microtome  in  the 
Journal  of  Applied  Microscopy,  Vol.  VI.  In  a  word,  it  is  now  possible  with 
the  almost  perfect  automatic  microtomes  to  make  thousands  of  perfect  sections 
where  in  1860  only  occasionally  could  the  most  expert  get  tens  with  his  hand 
sectioning. 

\  433.  Types  of  Microtomes. — There  are  two  great  types  :  (i)  The  early 
type  in  which  the  preparation  to  be  sectioned  is  held  mechanically  and  moved 
up  by  a  screw,  the  section  knife  being  held  in  the  hand  and  moved  across  the 
object  usually  with  a  drawing  motion  as  in  whittling  (Fig.  228). 

(2)  The  mechanical  type  in  which  both  specimen  and  knife  are  mechani- 
cally held  and  guided,  and  the  operator  simply  supplies  power  to  the  machine. 

In  the  highest  types  of  the  second  class — automatic  microtomes — the 
operator  only  needs  to  put  the  knife  and  specimen  in  position  and  supply  the 
power  and  sections  of  any  thickness  and  any  number  may  be  produced  in  a 
short  time.  A  skilled  and  experienced  person  can  get  better  results  here  as 
well  as  with  free-hand  sectioning  or  the  hand  microtome.  Even  automatic 
machines  work  better  for  skilled  workmen. 

As  is  seen  by  the  accompanying  cuts,  sometimes  the  knife  is  fixed  in  posi- 
tion and  the  object  to  be  sectioned  moves,  while  in  other  forms  the  object  to 
be  sectioned  remains  fixed  and  the  knife  moves.  Furthermore  for  sectioning 
paraffin,  the  knife  meets  the  object  like  a  plane  (straight  cut),  while  for  col- 
lodion sectioning  the  knife  is  set  obliquely  and  there  results  an  oblique  or 
drawing  cut  as  in  whittling. 

\  434.  Section  Knives. — A  section  knife  should  have  the  following  char- 
acters, (i)  The  steel  should  be  good.  (2)  The  blade  should  be  slightly  hol- 
low ground  on  both  sides.  Why  some  makers  persist  in  grinding  one  side 
flat  is  a  mystery.  (3)  The  edge  of  the  knife  should  be  straight,  not  curved  as 
in  a  shaving  razor.  (4)  The  back  should  be  parallel  with  the  edge.  (5)  The 
blade  should  be  long,  12  to  15  centimenters,  as  it  takes  no  more  time  or  skill 
to  sharpen  a  large  than  a  small  knife.  (6)  The  blade  should  be  heavy.  There 
was  formerly  a  fashion  of  making  very  thin  bladed  section  knives,  but  that  is 
a  great  mistake,  for  the  thin  blade  bends  and  vibrates  in  cutting  firm  tissue 
and  large  pieces.  There  is  no  possible  advantage  in  a  thin  bladed  section 
knife  for  microtome  work,  but  much  disadvantage  from  the  lack  of  rigidity. 


288  MICROTOMES  AND  SECTION  KNIVES  \_CH.  X 

The   microtome   knives   shown   on   the   various   instruments   figured   in   this 
chapter  illustrate  well  the  proper  form  of  section  knives.      (Figs.  227,  238.) 


FIG.  227.     Section  knife  with  the  honing  back  in  position  (Cut  loaned  by 
the  Spencer  Lens  Co. ) 

§  435-  Sharpening  Section  Knives  ;  Hones  and  Strops. — 
Perhaps  it  should  be  taken  for  granted  that  any  one  would  appre- 
ciate the  impossibility  of  making  good  sections  with  a  dull  section 
knife,  but  experience  teaches  the  contrary.  Students  are  prone  to 
believe  that  with  one  of  the  elaborate  automatic  microtomes,  good 
sections  may  be  made  with  any  kind  of  an  edge  on  the  knife.  It  is 
forgotten  that  the  knife  is  the  most  important  part,  all  the  other 
mechanism  is  simply  its  servant. 

For  sharpening,  select  a  fine,  yellow  Belgian  hone,  and  a  very 
fine  Arkansas  hone.  As  a  rule  hones  from  the  factory  are  not  suffi- 
ciently plane.  They  may  be  flattened  by  rubbing  them  on  a  piece 
of  plate  glass  covered  with  moderately  fine  emory  or  carborundum 
wet  with  water.  Round  the  corners  and  edges  of  the  hones  on  the 
plate  glass  or-  on  a  grindstone.  In  using  the  Belgian  hone  for 
sharpening  knives,  wet  the  surface  well  with  a  moderately  thick 
solution  of  soap.  With  the  Arkansas  stone  use  some  thin  oil — 
xylene  or  kerosene  mixed  with  a  little  olive  oil  or  machine  oil. 

Honing.  Before  honing  a  section  knife,  make  sure  that  the 
edge  is  smooth,  that  is  that  it  is  free  from  nicks.  Test  this  by 
shaving  off  the  surface  of  a  block  of  paraffin.  If  nicks  are  present 
the  cut  surface  will  show  scratches.  It  is  advisable  also  to  look  at 
the  edge  of  the  knife  with  a  magnifier  and  with  a  low  power  (50 
mm. )  objective.  If  nicks  are  are  present  remove  them  by  draw- 
ing the  edge  along  a  very  fine  Arkansas  hone. 


CfJ.X]  SECTIONING  289 

A  saw  edge  may  be  all  right  for  rough  cutting  and  for  shaving 
razors,  but  if  one  wishes  to  get  perfect  sections  2  to  io/*  in  thick- 
ness a  saw  edge  will  not  do.  In  removing  the  nicks  one  should 
of  course  bear  on  very  lightly.  The  weight  of  the  knife  is  usually 
enough. 

In  honing  use  both  hands,  draw  the  knife,  edge  foremost,  along 
the  hone  with  a  broad  curved  motion.  In  turning  the  knife  for  the 
return  stroke,  turn  the  edge  up,  not  down.  Continue  the  honing 
until  the  hairs  on  the  arm,  wrist  or  hand  can  be  cut  easily  or  until 
a  hair  from  the  head  can  be  cut  within  5  mm.  from  the  point  where 
it  is  held.  The  sharper  the  knife  becomes  the  lighter  must  one 
bear  on.  One  should  also  use  the  finest  stone  for  finishing.  If  one 
bears  on  too  hard  toward  the  end  of  sharpening,  the  edge  will  be 
filled  with  nicks. 

In  honing  and  stropping  large  section  knives,  there  has  come 
into  use  during  the  last  few  years  the  so  called  "  honing  backs". 
These  elevate  the  razor  slightly  so  that  the  wedge  is  blunter  and 
one  does  not  have  to  grind  away  so  much  steel,  (Fig.  227). 

Strop.  A  good  strop  may  be  made  from  a  piece  of  leather 
(horse  hide)  about  50  cm.  long  and  5  to  6  cm.  wide,  fastened  to  a 
board  of  about  the  same  size. 

The  strop  is  prepared  for  use  by  rubbing  into  the  smooth  sur- 
face some  carborundum  powder,  i.  e.  60  minute  carborundum,  that 
which  is  so  fine  that  it  remains  in  suspension  in  water  for  60 
minutes,  or  one  may  use  diamantine  or  Jewelers'  rouge. 

Stropping.  With  the  back  foremost  draw  the  knife  length- wise 
of  the  strop  with  a  broad  sweep.  For  the  return  stroke  turn  the 
edge  up  as  in  honing.  Continue  the  stropping  until  a  hair  can  be 
cut  i  to  2  centimeters  from  where  it  is  held. 

§  436.  Free-Hand  Sectioning. — To  do  this  one  grasps  the 
section  knife  in  the  right  hand  and  the  object  in  the  left.  Let  the 
end  to  be  cut  project  up  between  the  thumb  and  index  finger.  One 
can  let  the  knife  rest  on  the  thumb  or  index  finger  nail  and  with  a 
drawing  cut  make  the  section  across  the  end  of  the  piece  of  tissue. 
By  practice  one  learns  to  make  excellent  sections  this  way.  If  the 
whole  section  is  not  sufficiently  thin,  very  often  a  part  will  be  and 
one  can  get  the  information  needed. 

§  437.     Sectioning   with  a  Hand  or  Table  Microtome.— 


290 


SECTIONING 


[  CH.  X 


The  tissue  is  held  by  the  microtome  and  moved  up  by  means  of  a 
screw.  The  knife  rests  on  the  top  of  the  microtome  and  is  moved 
across  the  tissue  by  the  hand.  Microtomes  of  this  kind  are  excel- 
lent. No  one  need  wait  for  expensive  automatic  microtomes  to  do 
good  sectioning.  With  a  good  table  microtome  the  knife  being 
guided  by  the  hand  or  hands  of  the  operator,  he  can  make  straight 
cuts  as  for  paraffin  sectioning,  or  drawing  cuts  as  for  collodion  work. 
(Figs.  228-229). 


FIG.  228  FIG.  229 

FIGS.  228-229.  Hand  and  table  microtomes.  Both  have  a  screw  f 01  ele- 
vating the  object  to  be  cut  and  a  surface  on  which  to  rest  the  section  knife. 
228  in  held  in  the  hand,  229  is  fastened  to  a  table.  The  knife  is  held  and  moved 
by  the  hand  in  both  cases.  ( Cuts  loaned  by  the  Bauch  &  Lomb  Optical  Co) . 

§  438.  Sectioning  with  a  Freezing  Microtome. — In  this 
.method  of  sectioning  the  tissue  is  rendered  firm  by  freezing  and  the 
sections  are  cut  rapidly  by  a  planing  motion  as  with  paraffin.  Now 
the  most  usual  freezing  microtome  is  one  in  which  the  freezing  is 
done  with  escaping  liquid  carbon  dioxid.  The  microtome  is  in 
general  like  the  one  shown  in  Fig.  229.  The  knife  should  be  very 
rigid.  A  plane  blade  is  often  made  use  of.  The  tissue  may  be 
either  fresh  or  fixed.  If  alcohol  has  been  used  it  must  be  soaked 
out  of  the  tissue  by  placing  it  in  water.  Sometimes  tissues  are 


CM.  -V]  PARAFFIN  METHOD  291 

infiltrated  a  day  or  two  in  thick  mucilage  before  freezing.  Drop  a 
little  thick  mucilage  on  the  top  of  the  freezer,  put  the  tissue  in  the 
mucilage  and  turn  on  a  small  amount  of  carbon  dioxid.  It  will 
soon  freeze  the  mucilage  and  the  tissue  as  shown  by  the  white 
appearance.  When  frozen,  cut  the  tissue  rapidly.  It  is  well  to 
have  an  assistant  turn  the  feed  screw  up  while  the  sections  are  cut. 
When  20  or  30  sections  are  cut  place  them  in  water  or  normal  salt 
solution.  The  staining  and  mounting  of  the  sections  will  be  con- 
sidered in  §  461-471.  This  is  a  rapid  method  of  getting  sections 
much  used  in  pathology  where  quick  diagnoses  are  demanded.  In 
normal  histology  the  freezing  microtome  is  used  mostly  for  organs 
or  parts  of  greatly  varying  density.  For  example  if  one  wishes  sec- 
tions of  the  finger  and  finger  nail,  this  apparatus  offers  about  the 
only  means  of  getting  good  sections.  In  that  case  the  bone  is  decal- 
cified before  trying  to  make  the  sections  (§  398). 

THE   PARAFFIN   METHOD   OF  SECTIONING 

§  439.  Object  of  the  Paraffin. — In  the  early  periods  in  his- 
tology great  difficulty  was  encountered  in  making  good  sections  of 
organs  and  parts  of  organs  because  the  different  tissues  were  very 
unlike  in  density.  At  first  tallow  and  beeswax,  elder  pith,  liver 
and  various  other  substances  were  used  to  enclose  or  surround  the 
object  to  be  cut.  This  gave  support  on  all  sides,  but  did  not  render 
the  object  homogeneous.  In  the  early  sectioning,  a  great  effort 
was  made  to  keep  all  imbedding  material  from  becoming  entangled 
in  the  meshes  of  the  tissue.  This  was  guarded  against  by  coating 
the  object  with  mucilage,  and  hardening  it  in  alcohol.  This  muci- 
lage jacket  kept  the  tissue  free  from  infiltration  by  the  imbedding 
mass  and  itself  was  easily  gotten  rid  of  by  soaking  the  sections  in 
water. 

A  great  advance  was  made  when  it  was  found  that  the  imbed- 
ding mass  could  be  made  to  fill  all  the  spaces  between  the  tissue 
elements  and  surround  every  part,  the  tissue  assuming  a  nearly 
homogeneous  consistency,  and  cutting  almost  like  the  clear  imbed- 
ding mass.  Coco  butter  was  one  of  the  first  substances  to  be  used 
for  thus  "infiltrating"  the  tissues.  The  imbedding  mass  must  be 
removed  before  the  staining  and  mounting  processes. 

§  440.     Infiltration  of  the  Tissue  with  Imbedding  Mass.— 


292 


PARAFFIN  METHOD 


[OY.  X 


The  tissue  to  be  cut  in  this  way  is  first  fixed  by  one  of  the  fixers 
used  for  histology.  Several  good  ones  are  given  in  sections  406, 
416,  423,  429. 

(A)  The  tissue  is  then  thoroughly  dehydrated  by  means  of 
95%  and  absolute  alcohol.     For  most  objects,  especially  embryos 
and  other  colorless  objects  it  is  best,    during  the  dehydration,    first 
to  use  alcoholic  eosin  (§  402),  as  the  most  delicate  part  shows  when 
one  cuts  the  sections.     Leave  the  piece  of  tissue  to  be   cut  over 
night  in  alcoholic  eosin,  and  a  few  hours  in  uncolored  95%   alcohol 
using  20  times  as  much  alcohol  as  tissue.     For  the  final  dehydra- 
tion it  should  be  left  in  absolute  alcohol  four  or  five  hours  or  over 
night,  depending  on  the  size  of  the  object. 

(B)  Remove  the  alcohol  by  a  solvent  of  the  imbedding  mass, 
that  is  by  some  substance  which  is  miscible  with  both  alcohol  and 
the  imbedding  mass  (§  422,  441).     Cedar  wood  oil  is  most  generally 
used  (§  390).     Leave  the  tissue  in  cedar  oil  until  the  tissue  sinks 
and  the  thin  parts  of  the  specimen  become  translucent.     If  the  tissue 
does  not  sink  after  a  time  it  means  that  the  tissue  was  not   dehy- 
drated.    Of  course  this  does  not  apply  to  lung  or  other  spongy  tissue 
containing  mnch  air.     It  is  well  to  change  the  cedar  oil  once.     The 
used  cedar  oil  may  be  left  in  an  open  bottle  for  the  evaporation  of 
alcohol  and  used  over  and  over  again. 


A  B 

FIG.  230.  Paraffin  dish  for  infiltrating  in  the  Lillie  oven.  It  is  made  of 
copper  and  as  shown  has  a  handle  for  ease  in  transference.  A,  the  ivhole  dish, 
B,  the  dish  in  section.  (Jour.  Appl.  Micr.  1899,  p.  266. ) 

(C).     Displace  the  cedar  oil  by  melted  paraffin  wax.     When 
the  tissue  is  saturated  with  the  oil,  transfer  it  to  an  infiltrating  dish 


CH.  A'] 


PA R 'A WIN  METHOD 


293 


(Fig.  230),  containing  melted  paraffin.  Place  in  a  paraffin  oven 
(Fig.  231)  and  keep  the  paraffin  melted  for  from  two  hours  to  three 
days  depending  on  the  size  and  character  of  the  piece  to  be 
imbedded.  If  the  tissue  was  thoroughly  dehydrated  and  well 
saturated  with  cedar  oil  the  melted  paraffin  permeates  the  whole 
piece. 


FIG.  231.  The  Lillie  compartment,  paraffin  oven  for  infiltrating  tissues 
with  paraffin.  Various  sizes  of  this  arc  made  (8,  16  and  24  compartments}. 
Except  for  the  largest  laboratories  the  one  with  1 6  compartments  and  trays  will 
be  found  of  sufficient  capacity.  Dr.  Lillie  has  recently  omitted  a  part  of  the 
trays  and  thus  gained  compartments  for  receiving  dishes  in  which  paraffin  is 
kept  melted  and  ready  for  use.  (Cut  loaned  by  the  Spencer  Lens  Co. ) 

§  441.  Imbedding  in  Paraffin  Wax. — When  the  object  is 
thoroughly  infiltrated  imbed  as  follows  :  Make  of  strong  writing 
paper  a  box  considerably  larger  than  the  piece  to  be  imbedded. 


294 


PA  RA  FFIN  ME  THOD 


\_CH,  X 


Nearly  fill  the  box  with  paraffin  wax,  place  on  a  copper  heater 
(Fig.  241)  and  allow  to  remain  until  bubbles  appear  in  it.  Put  the 
box  on  cold  water  until  a  thin  stratum  of  paraffin  solidifies  on  the 
bottom.  Take  the  piece  of  tissue  from  the  melted  paraffin  (Fig. 
230)  and  arrange  in  the  box  for  making  sections  in  a  definite  direc- 
tion. Add  hot  paraffin  if  necessary,  and  then  place  the  box  on  cold 
water.  The  more  rapid  the  cooling  the  more  homogeneous  will  be 
the  block  containing  the  tissue  to  be  cut.  For  the  best  imbedding 
it  is  well  to  drop  95%  alcohol  on  the  surface  as  soon  as  a  film  has 
formed  in  cooling.  In  warm  climates  where  cold  water  is  not  easy 
to  procure  for  cooling  the  blocks,  one  may  float  the  paper  box  on 
95%  alcohol  and  with  a  pipette  (Fig.  240)  drop  strong  alcohol  on 
the  sides  of  the  box  and  on  the  top  of  the  paraffin  as  soon  as  a  sur- 
face film  has  formed. 

It  is  very  desirable  to  mark  on  the  box  the  name  of  the 
imbedded  object  and  to  indicate  which  end  or  face  is  to  be  cut.  See 
also  under  serial  sectioning  (§  472-473). 

FIG.  232.  Various  forms 
of  scalpels.  The  one  at  the 
left  is  especially  excellent  for 
cutting  the  ribbons  of  sec- 
tions of  the  proper  length  for 
mounting.  The  large  one 
with  straight  edge  is  the 
best  form  for  trimming  the 
paraffin  block  square  for 
sectioning.  {Cut  loaned  by 
the  Bausch  &  Lomb  Opti- 
cal Co.} 

§  442.  Fastening  the  Block  to  a  Holder. — Use  one  of  the 
block  holders  or  object  discs  furnished  with  the  microtome,  or  a 
short  stove  bolt  (Figs.  233-236).  Heat  the  larger  end  and  press 
the  paraffin  block  against  the  hot  metal  until  it  melts  the  paraffin. 
Hold  the  two  together  while  cold  water  flows  over  them.  When 
cold  the  block  is  firmly,  cemented  to  the  holder.  Pains  should  be 
taken  to  have  tne  axis  of  the  block  parallel  with  the  long  axis  of 
the  holder  ;  and  one  should  not  cut  the  block  so  short  that  the 
holder  comes  in  contact  with  the  tissue  when  the  two  are  cemented 
together. 

A  clamp  is  sometimes  used  for  holding  the  paraffin  block  (Figs. 
229,  246-247). 


en. 


PARAFFIN  METHOD 


295 


S  443.  Trimming  the  End  of  the  Block  for  Sectioning.— 
Sharpen  the  end  to  be  cut  in  a  pyramidal  form,  being  sure  to  leave 
2  millimeters  or  more  of  paraffin  over  the  tissue  at  the  end  as  well 
as  on  the  sides.  The  block  is  trimmed  in  a  pyramidal  form  so  that 
it  will  be  rigid.  Take  particular  pains  that  the  opposite  faces  at  the 
end  of  the  block  are  parallel,  and  all  the  corners  right  angles. 

S  444.  Making  Paraffin  Sections. — Put  the  paraffin  block 
or  the  metal  holder  in  the  clamp  of  the  microtome.  Arrange  the 
block  so  that  one  side  of  the  pyramidal  end  is  parallel  with  the  edge 
of  the  knife,  then  tighten  the  clamp,  and  if  an  automatic  microtome 
is  used  make  sure  that  the  section  knife  is  also  tightly  clamped  by 
the  proper  set  screws.  It  is  well  to  have  the  knife  lean  slightly 
toward  the  paraffin  block  (Fig.  239). 


FIG.  233.  The  Minot  automatic  rotary,  microtome  for  paraffin  sectioning 
(Sections  from  i  /.i  to  25  ft  may  be  cut  on  this  instrument. )  (Cut  loaned  by 
the  Bausch  &  Lomb  Optical  Co.). 


296 


PARAFFIN  METHOD 


\_CH.  X 


The  knife  edge  meets  the  paraffin  squarely  as  in  planing.  The 
thickness  of  section  is  provided  for  in  the  automatic  microtome  by 
the  indicator  which  may  be  set  for  any  desired  thickness,  or  one  can 
turn  up  the  screw  by  hand  in  the  table  microtome.  (Fig.  229). 
The  paraffin  and  its  contained  tissue  are  cut  in  a  thin  shaving.  If 
the  tissue  was  stained  in  toto  with  eosin  as  suggested  in  §  440  A,  it 
is  marked  out  with  great  clearness  in  the  containing  paraffin. 

As  succeeding  sections  are  cut  they  push  along  the  previous 
sections,  and  if  the  hardness  of  the  paraffin  is  adapted  to  the  tem- 
perature where  the  sectioning  is  done  the  edges  of  the  successive 
sections  will  be  soldered  as  they  strike.  This  produces  a  ribbon  as 
it  is  called,  and  if  the  paraffin  block  has  been  properly  trimmed  at 
the  end  the  ribbon  will  be  straight  and  even  (Fig.  234).  If  the  rib- 
bon is  curved  sideways  it  indicates  that  one  side  of  the  block  is 
thicker  than  the  other  and  the  sections  are  slightly  wedge  shaped. 


FIG.  234.  Automatic  rotary  microtome  for  paraffin  sectioning.  Sections 
from  if.i  to  ioot-1  may  be  cut  on  this  instrument.  (Cut  loaned  by  the  Spencet 
Lens  Co.) 

If  the  paraffin  is  too  hard  for  the  room  temperature  and  for  a 
given  thickness  of  section  the  sections  will  curl ;  if  it  is  too  soft  the 
sections  will  crumple. 


CIl.  X  ] 


PARAFFIN  METHOD 


297 


The  thinner  the  sections  the  harder  should  be  the  paraffin  or 
the  cooler  the  sectioning  room  ;  and  the  thicker  the  sections  and 
the  larger  the  object  to  be  cut,  the  softer  can  be  the  paraffin  and  the 
higher  the  temperature.  If  then  the  sections  do  not  ribbon,  make 
thinner  sections  or  work  in  a  warmer  place.  If  the  sections 
crumple,  make  thicker  sections  or  work  in  a  cooler  room.  Of  course 
one  can  reimbed  in  a  more  suitable  hardness  of  paraffin. 


Fi<;.  235.  The  Conklin-Pietzsch  automatic  lever  microtome  for  paraffin 
sectioning.  This  is  a  modified  Ryder  microtome  and  is  simpler  and  therefore 
cheaper  than  most  paraffin  instruments.  It  is  designed  for  sections  from 
/-joo  to  1 0-300  mm.  ( j  1-3  to  33  1-311 ) .  By  means  of  a  special  attachment, 
sections  of  211  may  be  made.  ( Cut  loaned  by  Edward  Pennock,  Philadelphia. ) 

In  the  season  when  steam  radiators  are  used  one  can  get  almost 
any  desired  temperature  by  sectioning  nearer  or  farther  from  the 
radiator. 


29S  PARAFFIN  METHOD  [  CH.  X 

In  the  winter  it  is  a  good  plan  to  warm  the  microtome  and  sec- 
tion knife  before  sectioning.  This  can  be  very  easily  done  by  put- 
ting a  cloth  over  the  radiator  and  the  microtome  something  like  a 
tent. 

|  445.  Electrification  of  the  Paraffin  Ribbons. — Some  days  there  is  such 
an  accumulation  of  static  electricity  in  cutting  the  ribbons  that  they  jump 
toward  anything  brought  near  them.  This  is  very  annoying  and  liable  to  be 
so  destructive  to  many  of  the  sections  that  serial  work  (|  472)  can  not  be  done 
with  safety. 

Many  devices  have  been  tried  to  overcome  this  difficulty,  like  burning  a 
gas  jet  near  the  microtome,  boiling  water  near  the  apparatus  etc. ,  but  the  safest 
way  is  to  wait  for  more  favorable  conditions. 

To  overcome  this  electrification,  Dixon,  (Jour.  Roy.  Micr.  Soc.,  1904,  p. 
590),  recommends  fastening  a  5  milligram  tube  of  radium  bromide  on  the 
knife  near  where  the  sectioning  is  done.  The  radium  ionizes  the  air  and  ren- 
ders it  a  good  conductor,  and  then  the  static  electricity  cannot  accumulate.  I 
have  not  been  able  to  try  this  method. 

|  446.  Storing  Paraffin  Ribbons. — The  most  convenient  method  for  caring 
for  the  ribbons  as  they  are  cut  is  to  place  them  on  a  tray  (Fig.  212)  lined  with 
a  sheet  of  white  paper.  It  is  important  to  write  on  the  paper  full  data,  giving 
the  name  of  the  tissue,  the  thickness  of  the  sections,  the  date  etc.  It  is  well 
also  to  number  the  ribbons  and  to  indicate  clearly  the  position  of  the  first 
section  or  the  beginning  of  the  ribbon. 

Ribbons  of  sections  on  a  tray  should  be  covered  by  another  tray  if  one 
wishes  to  carry  them  to  another  room.  The  slightest  gust  of  air  sends  them 

flying- 
Ribbons  on  trays  may  be  kept  a  long  time,  three  or  four  years  at   least,   if 
they  are  stored  in  a  cool  place.     The  sections  do  not  flatten  out  quite  as  well 
after  standing  a  long  time  as  they  do  soon  after  they  are  made. 

§  447.  Spreading  the  Sections. — Paraffin  sections  are  almost 
invariably  slightly  wrinkled  or  folded  in  cutting.  To  remove  the 
wrinkles  one  takes  advantage  of  the  expansion  of  paraffin  when  it  is 
warmed.  The  sections  may  be  floated  on  warm  water  when  they 
will  straighten  out  and  become  smooth,  or  the  usual  method  is  to 
'stretch  them  on  the  slide  upon  which  they  are  to  be  finally  mounted. 

§  448.  Spreading  Sections  on  a  Slide.  — A  double  operation 
is  performed  in  this  way,  viz;  the  sections  are  made  smooth  and  they 
are  also  fastened  to  the  slide.  Put  a  minute  drop  of  albumen  fixi- 
tive  on  the  middle  of  a  slide  (Fig.  187)  and  with  the  ball  of  one  fin- 
ger spread  it  over  the  slide,  making  a  thin  even  layer.  It  cannot  be 
too  thin.  It  is  liable  to  stain  if  it  is  too  thick. 


CH.  X  ] 


PARAFFIN  METHOD 


299 


A 


Fi<;.  236 


tr 


FIG.  237  A 


FIG.  237  B 


FIG.  238 


FIG.  239 


300 


[  CH.  X 


FIG.  236  A  B.  A  paraffin  holder  clamp  and  a  razor  support  for  the 
Jllinot  Microtome  for  utilizing  most  of  the  cutting  edge.  (  Trans.  Amer. 
Micr.  Soc.,  1901.) 

Clamp  for  the  paraffin  block  holder.  In  A  it  is  shoivn  in  section,  in  a 
side  view.  With  this  clamp  one  can  use  stove  bolts  as  well  as  the  expensive 
paraffin  holders  furnished  with  the  instrument.  A  laboratory  can  have  as 
many  paraffin  block  holders  as  necessary  without  undue  expense. 

FIG.  237  A  B.     Razor  Support  and  Razor. 

(A)  Support  with  heavy  base  and  vertical  piece.  The  base  should  be 
capable  of  moving  endwise  one  or  two  centimeters  to  bring  the  opening  in  the 
vertical  part  opposite  the  paraffin  block.  (B)  Front  piece  to  the  razor. 

FIG.  238.  Razor  with  straight  back  and  edge.  By  moving  this  back  and 
forth  on  the  support  nearly  the  entire  cutting  edge  can  be  utilized. 

FIG.  239  A  B.  The  knife  support  of  the  microtome  with  the  razor  support 
and  razor  in  position. 

(A)  Front  view  ;  (B}  Back  view,  in  which  the  inclination  of  the  knife 
tozvard  the  paraffin  block  is  shown. 

With  a  pipette  (Fig.  240)  put  several  drops  ot  water  on  the 
slide  and  then  place  a  piece  of  a  ribbon  on  the  water;  or  put  the  sec- 
tions on  the  albumenized  slide  and  add  the  water  afterward.  Heat 


FIG.  240.  Reagent  bottle  with  combined  cork  and  pipette 
( This  is  made  by  taking  a  cork  of  the  proper  size  and  making 
in  it  a  hole  with  a  cork  borer  for  the  glass  tube.  It  is  advan- 
tageous to  have  a  string  tied  tightly  around  the  rubber  bulb  as 
shown) . 


the  slide  carefully  over  a  spirit  lamp  or  gas  flame,  being  sure  not 
to  melt  the  paraffin.  As  the  water  warms  the  paraffin  expands  and 
stretches  the  sections  out  smooth.  A  copper  heating  plate  also, 
Fig.  241,  is  excellent  for  spreading  sections. 


CH.  X  ] 


301 


After  the  sections  are  spread,  drain  off  most  of  the  water,  arrange 
the  sections  with  a  needle  or  scalpel  and  place  the  slide  in  one  of  the 
trays  (Fig.  212).  Allow  it  to  remain  over  night  or  preferably  longer. 
The  longer  the  drying  in  air  the  more  surety  do  the  sections  adhere 
to  the  glass  slide. 


FIG.  241 


FIG.   242 
An  alcohol  or  small  Bunsen 


FIG.  241.  Copper  wanning  plate  on  legs, 
lamp  is  used  with  this.  It  is  more  satisfactory  to  spread  sections  by  warm- 
ing the  slides  on  this  plate  than  to  heat  them  directly  over  the  flame.  (Cut 
loaned  by  the  Spencer  Lens  Co). 

FIG.  242.  Spirit  lamp.  This  is  of  glass  and  has  the  sides  flattened  so 
that  the  lamp  rests  on  one  of  the  sides  if  it  is  overturned.  (Cut  loaned  by  the 
Bausch  &  Lomb  Optical  Co.). 

If  one  is  in  haste  to  take  the  succeeding  steps  in  the  preparation, 
the  slide  may  be  dried  by  putting  it  into  a  drying  oven  at  38°  1040° 
C.  for  half  an  hour  or  more.  The  slower  drying  in  air  is  better  if 
one  has  plenty  of  time. 

Some  tissues  are  very  difficult  to  get  perfectly  smooth  as  just 
described.  If  fine  wrinkles  persist  one  can  sometimes  overcome  the 
difficulty  by  letting  the  slide  cool  and  then  covering  with  a  piece  of 
fine  tissue  paper  slightly  moistened  ;  press  down  firmly  with  the  ball 
of  the  finger  on  the  sections.  Then  take  hold  of  the  edge  of  the 
paper  and  roll  it  off  the  sections.  Unless  one  is  careful  the  sections 
are  liable  to  come  away  with  the  paper  instead  of  adhering  to  the 
slide. 

As  the  water  dries  out  the  spread  sections  come  in  very  close 
contact  with  the  glass  and  adhere  quite  firmly  to  it.  The  thinner 
the  sections  the  more  tightly  do  they  stick.  This  makes  it  possible 
to  perform  the  rest  of  the  operations  on  the  slide.  One  has  to  be 
careful  not  to  let  strong  currents  strike  the  sections. 


302 


PARAFFIN  METHOD 


[  CH.  X 


§  449.  Deparaffining  in  Xylene. — This  is  accomplished  by 
using  a  solvent  of  paraffin.  The  best  and  safest  one  to  use  in  a 
laboratory  is  xylene.  Benzine,  gasoline,  and  even  kerosene  are 
used,  but  xylene  is  a  powerful  solvent  of  paraffin,  does  not  injure 
the  tissue,  and  is  not  very  inflammable,  due  to  the  large  amount  of 
carbon  in  its  molecule  (see  §  392). 

It  requires  only  a  few  minutes  to  dissolve  paraffin  from  the  sec- 
tions, but  a  day  or  more  in  the  xylene  does  no  harm. 

When  the  paraffin  is  removed  the  staining  and  other  operations 
necessary  for  a  completed  preparation  may  be  undertaken  (See  for 
these  §  461-471). 


FIG.   243  FIG.  244 

FIG.  243.     Slide  basket  or  holder  and  a  glass  stoppered  bottle  to  contain 

the  same.  Xylene  is  safer  than  benzin  for  deparaffining.     The  slide  basket  was 

devised  by  Dr.  A.  B.  Mix  in  the  author' slab  oratory  in  1898.     It  is  cylindrical 

and  has  an  unjointed  handle  or  bail.     (Jour.  Ap.  Micr.,  vol.  i,  s8o8,  p.  s6o). 

FIG.  244.  Square  slide  basket  with  hinged  bail  or  handle  so  that  it  may 
tbe  turned  down  in  inserting  or  removing  the  slides.  In  1900  the  hinged  bail 
was  added  to  the  round  slide  baskets,  and  in  1902  the  form  was  changed  from 
round  to  square.  The  square  form  is  more  convenient,  and  suitable  for  all 
sizes  of  slides.  ( Cut  loaned  by  the  Spencer  Lens  Co. ) . 

§  450.  Collodionizing  the  Sections. — Except  for  carmine 
stains  and  perhaps  some  others,  collodion  remains  practically  color- 
less. While  the  sections  remain  quite  firmly  attached  to  the  slide 
after  they  have  been  spread  and  dried,  thick  sections  are  liable  to 


CH.  X] 


PARAFFIN  METHOD 


303 


come  off  in  the  many  processes  of  staining,  and  if  one  has  many 
sections  on  a  slide  some  of  them  may  become  loosened.  Tp  avoid 
this  the  sections  are  covered  with  a  delicate  layer  of  collodion, 
which  holds  them  down  to  the  slide.  The  early  method  was  to  use 
a  soft  brush  and  paint  a  thin  film  over  the  dried  sections  before  they 
were  deparaffined.  Now  the  sections  are  deparaffined,  and  then 
after  draining  the  xylene  from  the  slide,  10-15  seconds,  it  is  put 
into  a  bottle  containing  y\%  collodion  (§  395).  In  a  minute  or 
more  the  collodion  displaces  the  xylene  and  penetrates  the  sections 
and  forms  a  delicate  veil  over  their  free  surface.  No  harm  is  done 
by  leaving  the  sections  in  the  collodion  a  considerable  time,  but  a 
minute  or  two  is  sufficient.  The  slide  is  removed,  allowed  to  drain 
for  half  a  minute,  and  then  put  into  a  jar  of  67%  alcohol  (Fig.  222). 
The  alcohol  fixes  the  collodion  and  removes  the  ether.  As  the  67% 
alcohol  does  not  hurt  the  tissue  it  may  stay  in  the  jar  a  day  or  more 
if  desired,  but  half  an  hour  suffices. 

Steps  in  Order  for  the  Paraffin  Method. — §  439,  450,   461- 
471. 


Name 


No. 


Animal 

Date ;' 

Fixer 

Time  of  fix 

Washed  in  water 

67",,  ale 82%  ale 

Decalc.  $  398 67,  82%  ale.. 

In  toto  stain 

Washed  in 

67",,  ale 82%  ale 

95",,  ale.  and  eosin 


Absl.  ale. Cedar  oil.. 

Infill. 

Temp,  bath Imbed,  in. 

Sections  cut //'s 

Temp,  room 

Stains __ 


Mtd.  in 

Remarks  . 


3o4  COLLODION  METHOD  [  CH.  X 

The  sections  are  now  ready  for  the  subsequent  staining  and 
other  operations  to  make  a  finished  slide.  One  has  to  remember 
that  if  mucicarmin  (§  389)  is  to  be  used  in  staining,  the  prepara- 
tion must  not  be  collodionized  as  carmin  stains  collodion. 

THE    COLLODION    OR    CELLOIDIN    METHOD    OF   SECTIONING 

§  451.  Collodion  Method.  —  In  this  method  the  tissue  is 
thoroughly  permeated  with  a  solution  of  collodion  which  is  after- 
ward hardened.  Unlike  the  paraffin  of  the  paraffin  method,  the 
collodion  is  not  subsequently  removed  from  the  tissue,  but  always 
stays  in  the  sections.  It  is  transparent  and  does  no  harm. 

The  fixing  and  dehydration  with  95%  alcohol  is  the  same  as 
for  the  paraffin  method  (§§  430,  440). 

The  paraffin  method  gives  thinner  sections  than  the  collodion 
method  and  for  series  and  large  numbers  of  sections  is  superior. 

The  collodion  method  requires  no  heat  for  infiltration,  and  it 
does  not  render  the  firmer  forms  of  connective  tissue  so  hard  and 
difficult  to  cut.  It  is  especially  adapted  for  making  sections  of 
large  pieces  of  tissue  or  organs  and  when  thick  sections  are  desired. 
It  is  not  easy  to  cut  sections  less  than  10  /*  with  collodion,  while 
with  paraffin  it  is  possible  to  make  good  ribbons  of  small  objects  of 
delicate  texture  2  yu  to  3  //  in  thickness.  With  a  very  sharp  knife 
and  small  delicate  object,  and  one  of  the  better  forms  of  microtomes 
one  can  cut  short  series  in  i  //  sections  and  get  perfect  ribbons. 

Collodion  sectioning  is  sometimes  denominated  the  "  wet 
method  '  '  as  the  tissue  and  sections  must  always  be  wet  with  some 
liquid,  while  the  paraffin  method  is  called  the  "  dry  method"  as  the 
tissue  once  infiltrated  with  paraffin  keeps  in  the  air  indefinitely  and 
in  cutting  the  sections  no  liquid  is  used. 

§  452.  Infiltration  with  Ether  Alcohol.  —  Transfer  the 
piece  of  tissue  to  be  cut  from  95%  alcohol  to  a  mixture  of  equal 
parts  of  sulfuric  ether  and  95%  alcohol  and  leave  in  this  for  a  few 
hours  or  a  day  or  more  as  is  most  convenient.  This  is  to  soak  the 
tissue  full  of  a  solvent  of  the  collodion. 


§  453.  Infiltration  with  \%%  Collodion.—  Pour  off  the 
ether-alcohol  from  the  tissue  and  add  il/>%  collodion.  Leave  in 
this  over  night  or  longer  if  the  piece  of  tissue  is  large. 


(  H  .  \  'I  COLLODION  METHOD  305 


S  454.     Infiltration  with  3%  Collodion.—  Pour  off  the  i 
collodion  and  put  in  its  place  3/0    collodion.     Leave    the   tissue    in 
this  half  a  day  or  longer. 

$  455.  Infiltration  with  6%  Collodion.  —  Pour  off  the  3% 
and  add  6%  collodion  to  the  piece  of  tissue.  For  complete  infiltra- 
tion with  this  thick  collodion  leave  the  tissue  in  it  for  one  day  at 
least.  If  the  object  is  large  it  is  advantageous  to  leave  it  in  for  a 
week  or  two. 

FKV.  245.     Slender  dish  for  hardening  the  collodion   in  ^S          H^> 
chloroform  or  in  alcohol.     (Cut  loaned  by  the  Whitall,   Tatum   ffl^Bi 
Co.}. 

§  456.  Infiltration  and  Imbedding  in  8%  Collodion.  —  Pour 
off  the  6%  and  add  8%  collodion.  Leave  the  tissue  in  this  at  least 
one  day,  and  as  much  longer  as  possible  up  to  two  or.  three  weeks 
if  the  piece  of  tissue  is  large. 

(A)  For  imbedding  small   pieces   use  a  piece  of  wood,    (deck 
plug),  vitrified  fiber,  glass  or  a  good  cork  for  a  holder  and  cover  the 
end  with  6%  collodion  and  let  it  get  well  set  in  the  air,  then  put 
the  piece  of  tissue  on  the  holder  and  drop  8%  collodion  upon   it   at 
intervals  until  it  is  well  covered  all  around.     If  one  takes  consider- 
able time  for  this  the  collodion  thickens  greatly  in  the  air.     This  is 
an  advantage  for  it  gives  a  denser  block  for  sectioning.     After  the 
collodion  is  pretty  well  set,  place  holder  and  tissue  in  a   vessel  with 
chloroform  to  harden.     One  can  put  the  preparation  into  the  chloro- 
form or  if  the  vessel  is  tight  it  may  be   above   the   chloroform,    the 
vapor  then  acting  as  the  hardener. 

(B)  Imbedding  in  a  box.  —  If  the  object  is  of  considerable  size 
it  is  best  to  use  a  paper  box  for  imbedding  as    with   paraffin.     If   a 
very  small  amount  of  vaseline  is  rubbed  on  the  inside  of  the  box    it 
prevents  the  collodion  from  sticking  to  the  paper. 

Put  first  some  of  the  8%  collodion  in  the  box  and  let  it  remain 
in  the  air  until  nearly  solid,  2  to  3  minutes.  Then  arrange  the 
specimen  to  be  cut  as  for  imbedding  in  paraffin,  and  add  gradually 
8%  collodion  until  the  object  is  well  covered.  Let  the  box  stand 
for  a  few  minutes  in  air,  then  place  it  in  a  dish  like  a  Stender  dish 
(Fig.  245)  and  pour  some  chloroform  on  the  bottom  of  the  dish. 
Cover  and  the  collodion  will  harden  partly  by  the  chloroform  vapor 


3o6  COLLODION  METHOD  \_CH.X 

and  partly  by  that  which  soaks  through  the  paper.  It  is  well  to 
change  the  chloroform  at  least  once.  The  used  chloroform  will  con- 
tain some  ether- alcohol,  but  is  good  for  killing  animals. 


FIG.  246.  Microtome  for  collodion  sectioning.  A  microtome  of  this  form 
may  also  be  used  for  paraffin  sectioning.  In  that  case  the  knife  is  set  at  right 
angles  in  order  to  cut  the  block  square  across  instead  of  ivith  a  drawing  cut  as 
for  collodion.  (Cut  loaned  by  the  Bausch  &  Lomb  Optical  Co.). 

After  24  or  48  hours  the  collodion  should  be  firm  all  through. 
Then  it  is  placed  in  67%  alcohol  where  it  may  be  left  a  day  or 
more.  If  it  is  to  be  left  an  indefinite  time  the  67%  alcohol  should 
be  changed  for  82%. 

§  457-  Sectioning  by  the  Collodion  Method. — For  this 
one  can  use  a  table  microtome  (Fig.  229)  or  one  of  the  sliding 
microtomes  (Figs.  246,  247).  The  sections  are  made  with  a  knife 
set  obliquely  and  hence  with  a  drawing  cut. 

The  holder  with  the  small  piece  of  tissue  is  clamped  in  the 
microtome  and  arranged  as  desired,  then  the  sections  are  made  with 
an  oblique  knife  which  is  kept  wet  with  82%  alcohol.  The  best 
way  to  keep  the  knife  wet  is  to  have  a  dropping  bottle  over  the 


CH.  -V] 


COLLODION  I\fETHOD 


3°7 


object,  the  drops  falling  about  every  two  seconds.  As  the  sections 
are  cut  they  are  drawn  up  towards  the  back  of  the  section  knife 
with  a  soft  brush.  They  can  be  kept  in  order  in  this  way  and  not 
interfere  with  succeeding  sections. 

Some  operators  in  drawing  the  knife  across  the  tissue  use  a 
slight  sawing  motion.  However  one  proceeds,  the  knife  is  drawn 
rather  slowly,  not  rapidly  as  with  paraffin  work. 


FIG.  247.  Pietzsch  microtome^  University  of  Pennsylvania  model.  The 
knife  is  set  very  obliquely  for  collodion  sectioning.  For  paraffin  sectioning  the 
knife -Mould  be  at  right  angles  to  the  clamp.  (Cut  loaned  by  Edward  Pen- 
nock,  Philadelphia.') 

If  the  imbedding  was  done  in  a  paper  box,  remove  the  box 
and  trim  the  collodion  block  suitably.  Dry  the  end  away  from  the 
tissue,  wet  it  with  3%  collodion.  Use  a  piece  of  wood,  a  cork  or 
other  holder  of  suitable  size.  Put  some  6%  collodion  on  the  holder 
and  let  it  dry  for  a  minute  or  so,  then  press  the  collodion  block 
down  on  the  holder.  Leave  in  the  air  for  a  minute  or  two  and 
then  put  into  67%  alcohol  to  harden  the  cementing  collodion. 


308  COLLODION  METHOD  \_CH.X 

After  15  minutes,  or  longer  if  convenient,  put  the  mounted  speci- 
men into  the  clamp  of  the  microtome  and  cut  as  above. 

Sometimes  when  the  imbedded  object  is  of  sufficient  size  and 
the  collodion  block  is  firm,  the  block  itself  is  put  into  the  micro- 
tome clamp,  no  wood  or  cork  holder  being  used. 

§  458.  Transferring  Sections  from  the  Knife  to  the 
Slide. — When  one  has  cut  the  number  of  sections  for  one  slide  they 
should  be  transferred  to  the  slide  as  follows  :  Take  a  piece  of 
white  tissue  paper  about  3x6  centimeters  in  size  and  lay  it  on  the 
knife  over  the  sections.  Press  down  slightly  so  the  paper  is  in 
contact  with  all  the  sections.  Take  hold  of  the  paper  beyond  the 
edge  of  the  knife  and  gradually  pull  it  down  off  the  knife. 

If  there  is  the  right  amount  of  alcohol  on  the  knife  the  sections 
adhere  to  the  paper  and  move  with  it.  This  transfers  the  sections 
from  the  knife  to  a  piece  of  tissue  paper.  Place  the  tissue  paper 
with  the  sections  down  on  the  middle  of  an  albumenized  slide. 
Cover  with  another  piece  of  paper  and  press  down  gently.  This 
presses  the  sections  against  the  slide  and  absorbs  a  part  of  the 
alcohol.  Take  hold  of  one  edge  of  the  paper  and  lift  it  with  a  roll- 
ing motion  from  the  slide.  The  sections  should  stay  on  the  slide. * 

§  459.  Fastening  the  Sections  to  the  Slide. — With  a 
pipette,  drop  95%  alcohol  on  the  slide  of  sections,  then  use  a 
pipette  full  of  absolute  alcohol  if  it  is  at  hand.  Drain  most  of  the 
alcohol  away  and  add  a  few  drops  of  ether-alcohol.  The  collodion 
should  melt  and  settle  down  closely  on  the  slide.  If  the  collodion 
does  not  melt  the  dehydration  was  not  sufficient  and  more  alcohol 
must  be  used.  After  the  collodion  has  melted  down  upon  the  slide 
let  the  slide  remain  a  minute  or  two  in  the  air,  and  then  transfer 
the  slide  to  ajar  of  67%  alcohol.  (Figs.  243,  251.) 

After  half  an  hour  or  longer  the  preparation  is  ready  to  stain, 
etc.  See  below  for  directions  (§  §  461-471). 


*  Various  forms  of  paper  have  been  used  to  handle  the  collodion  sections. 
It  should  be  moderately  strong,  fine  meshed  and  not  liable  to  shed  lint,  and 
fairly  absorbent.  One  of  the  first  and  most  successful  papers  recommended  is 
"  closet  or  toilet  paper."  Cigarette  paper  is  also  excellent.  In  my  own  work 
the  heavy  white  tissue  paper  has  been  found  almost  perfect  for  the  purpose. 
Ordinary  lens  paper  or  thin  blotting  paper  for  absorbing  the  alcohol  or  oil 
may  be  used  with  it. 


CH.  A'] 


COLLODION  MKTIIOD 


3<>9 


FIG.  248.  Waste  bowl  with  rack  for  supporting  slides  and  a  small  funnel 
in  which  the  slides  stand  while  draining.  This  outfit  is  easily  made  by  any 
tinsmith.  The  rack  is  composed  of  two  brass  rods  about  4  mm.  in  diameter. 
The  be>it  end  pieces  are  sheet  lead.  The  funnel  is  made  of  tin,  copper  or  brass. 
Either  copper  or  brass  is  preferable  to  tin.  A  glass  dish  like  that  shown  in 
Figs.  /SS,  251  is  better  than  a  bowl,  as  it  can  be  more  readily  and  thoroughly 
cleaned.  (Cut  loaned  by  Wm  Wood  &  Co.] 

\  460.  The  Castor-Xylene  Method  of  Sectioning. — The  preparation  of  the 
tissue  is  the  same  as  described  in  \  451-456,  except  that  when  the  collodion  is 
hardened  in  chloroform  it  is  transferred,  not  to  alcohol,  but  the  block  is  placed 


FIG.  249.  Perforated  section  lifter.  This  is  easily  made  by  soldering  a 
wire  to  some  very  thin  sheet  brass  or  copper,  and  then  perforating  this  with  a 
coarse  needle  or  fine  awl.  Any  roughness  must  be  removed  by  using  a  fine 
oil  stone. 

in  Castor-Xylene  (§  392).-  In  a  few  days  the  collodion  gets  as  transparent  as 
glass  and  one  can  see  the  tissue  within  with  great  clearness.  It  can  remain  in 
the  castor-xylene  indefinitely. 


STAINING  AND  PERMANENT  MOUNTING       [  CH.  X 


In  cutting  one  proceeds  exactly  as  in  \  457  except  that  the  block  is  kept 
wet  with  castor- xylene  and  not  with  alcohol.  The  sections  are  arranged  on 
the  knife  and  transferred  to  the  slide  in  the  same  way  as  for  alcohol  section- 
ing (§  457-458). 

For  fastening  the  sections  to  the  slide,  as  no  water  is  present,  one  can  add 
the  ether-alcohol  at  once.  It  is  advantageous  here  to  have  a  mixture  of  ether 
2  parts  and  absolute  alcohol  one  part  for  melting  the  collodion  in  these  oil 
sections. 

Allow  the  slide  to  remain  in  the  air  till  the  collodion  begins  to  look  dull, 
then  the  slide  may  be  transferred  to  a  jar  of  xylene  to  remove  the  oil.  From 
the  xylene  it  is  transferred  to  95%  alcohol  and  then  the  slide  is  ready-  to  be 
stained,  etc.  as  described  below  (§  461-471). 


Steps  in  Order  for  the  Collodion  Method. — \  451-460,  461-47  r. 


Name 


No. 


Animal 95%  ale... 

Date Ether-ale. 

Fixer  

Time  of  fix 

Washedin  water 

67%  ale 82%  ale 

Decalc.  \  398 

67%  ale 82%  ale 

In  toto  stain 

Washed  in 

I 

67%  ale 82%  ale i   Remarks 

i 

STAINING    AND    PERMANENT    MOUNTING 

\  461.  Generalities  on  Stains. — From  the  standpoint  of  the  object  to  be 
stained,  dyes  may  be  divided  into  two  great  groups  : 

(i)  (a)  Those  which  select  out  or  differentiate  certain  parts  of  the  tissue 
and  make  them  prominent.  Such  dyes  are  called  then  differential  or  selective. 
If  the  nucleus  is  the  part  selected,  the  dye  is  frequently  called  a  nuclear  dye. 

(by  General  or  counter  stains.     These  stain  all  parts  of  the  tissue,  and  are 


6%  col 8%  col 

Imbedded 

Chloroform 67%  ale 

Or  castor-xylene 

Sections   cut /i's 

Stains 

Mounted  in__ 


CH.  X]       STAINING  AND  PERMANENT  MOUNTING  311 

usually  contrasting  in  color  ;  blue  or  purple  and  bright  red  are  frequent  com- 
binations, e.  g.  hematoxylin  and  eosin. 

(2)  From  the  standpoint  of  the  solvent  used  in  preparing  the  stains  they 
are  called  (a)  sit/neons,  and  (b)  alcoholic. 

If  one  uses  an  aqueous  stain  the  object  must  be  well  wet  with  water  before 
the  stain  is  applied,  and  afterward  well  washed  with  water  before  put  again 
into  alcohol.  If  an  alcoholic  stain  is  used  the  object  to  be  stained  should  be 
from  alcohol  of  the  same  strength  as  that  used  in  making  the  dye.  The  dye  is 
also  washed  away  from  the  tissue  with  the  same  strength  of  alcohol ;  it  may 
then  be  put  into  the  stronger  alcohols  for  dehydration. 

For  other  classifications  of  dyes  consult  the  larger  works,  Lee,  Mann, 
Ehrlich,  and  the  microscopical  journals. 


FIG.  250.  Pipette  with  large  rubber  bitlb  for  adding  liquids  to  prepara- 
tions. (Cut  loaned  by  the  Bausch  &  Lomb  Optical  Co. ) 

\  462.  Generalities  on  Mounting. — For  permanent  preparations  one  can 
use  a  medium  like  glycerin  or  glycerin  jelly  etc.  which  mixes  with  water. 
The  method  of  procedure  is  given  in  \  407,  408. 

For  the  most  permanent  mounting  resinous  media  are  used,  and  of  these 
resinous  media  Canada  balsam  (§  383)  has  been  longest,  and  is  now  most  used. 

In  mounting  in  balsam  one  must  remember  the  fundamental  principles  : 
(i )  the  object  to  be  mounted  in  balsam  must  not  contain  water.  It  must  then 
be  dried  or  desiccated,  or  it  must  be  rendered  anhydrous  by  some  liquid  which 
mixes  with  water.  As  all  tissues  and  organs  contain  much  water,  to  mount 
them  in  balsam  without  drying  in  the  air,  which  would  spoil  them  in  most 
cases,  one  must  take  the  following  steps  ( i )  Dehydrate  by  alcohol  which  mixes 
with  and  displaces  the  water;  (2)  Displace  the  alcohol  by  some  liquid  which 
mixes  with  it  and  is  also  miscible  with  balsam,  e.  g.  xylene,  etc.  (§  392). 
(3)  As  the  liquid  used  just  before  the  balsam  usually  makes  the  tissue  more  or 
less  translucent  it  is  often  called  a  "clearer".  Finally  displace  the  xylene  etc. 
by  balsam.  If  all  the  water  is  not  removed  in  some  way,  the  specimen  will 
look  turbid.  If  there  is  but  a  trace  of  water  present  and  one  uses  natural 
balsam  (?  383  )  for  mounting  the  small  amount  of  water  will  finally  disappear; 
but  it  is  better  to  dehydrate  the  tissue  thoroughly  before  adding  the  balsam. 

HEMATOXYLIN   WITHOUT    AND    WITH    COUNTERSTAINING 

^  463.  Staining  with  Hematoxylin. — Take  a  slide  of  sections 
prepared  by  the  paraffin  or  the  collodion  method  (§  450,  459  )  from 
the  jar  of  alcohol  and  plunge  it  into  a  vessel  of  water  to  remove  the 
alcohol.  For  staining  put  the  slide  of  sections  into  a  jar  or  shell 


3I2 


STAINING  AND  PERMANENT  MOUNTING       [  CH.  X 


vial  of  the  hematoxylin  solution  (Figs.  243,  251  )  or  one  can  lay 
the  slide  flat  on  the  staining  rack  or  some  other  support  and  add  the 
stain  to  the  sections  (Fig.  248  ).  It  usually  takes  from  2  to  10 
minutes  to  stain  sufficiently  with  hematoxylin.  A  good  plan  when 
one  is  learning  the  process  is  to  wash  off  the  stain  after  i  minute 
either  with  a  pipette  (Fig.  250  )  or  by  putting  the  slide  in  a  dish  of 
water.  Wipe  off  the  bottom  of  the  slide  and  put  it  under  the  micro- 
scope. Light  well,  use  a  low  power  and  one  can  see  the  nuclei 
stained  a  bluish  or  purple  color  as  hematoxylin  is  a  nuclear  dye.  If 
the  color  is  faint,  continue  the  staining  until  the  nuclei  stand  out 
boldly.  Sometimes  it  takes  a  long  time  to  stain  well  with  hematox- 
ylin. In  such  a  case  the  jar  of  stain  may  be  put  into  the  paraffin 
oven  and  the  heat  will  accelerate  the  staining.  One  may  also  heat 
the  individual  slides  as  for  spreading  sections,  but  one  must  be  care- 
ful not  to  let  the  stain  dry  on  the  sections.  As  the  stain  evaporates 
add  fresh  stain  with  a  pipette. 


FIG.  251.  Apparatus  and  regents  with  which  the  slide  holders  are  used. 
With  this^apparalus  it  is  easy  to  prepare  -specimens  in  large  numbers  very 
expeditiously.  After  the  sections  are  fastened  to  the  slide  and  placed  in  the 
holder,  the  slides  need  not  be  touched  during  all  the  operations  until  they  are 
finally  ready  to  be  mounted  in  balsam.  Each  holder  contains  from  12  to  14 
slides.  The  bottles  for  the  reagents  are  glass  stoppered  specimen  or  museum 
bottles.  (Mix,  Jour.  Ap.  Micr.  1898,  p.  777.) 

When  the  sections  are  well  stained  with  hematoxylin,  wash  off 
the  hematoxylin  with  water.  If  the  slide  is  allowed  to  stand  some- 
time in  ordinary  water  the  color  is  likely  to  be  brighter.  This  is 
due  to  the  action  of  the  alkali  (  ammonia,  etc.  )  usually  present  in 


CH.  X]       STAINING  AND  PERMANENT  MOUNTING  313 

natural  waters.  One  could  use  distilled  water,  adding  a  few  drops 
of  a  saturated  solution  of  lithium  carbonate. 

Dehydrate  in  95%  alcohol  and  absolute  if  necessary  ;  clear  and 
mount  in  balsam  as  described  in  the  next  section  (§  464). 

Hematoxylin  is  so  nearly  a  pure  nuclear  stain  for  most  tissues 
and  organs  that  the  cell  bodies  are  not  very  evident  with  this  alone, 
hence  some  counter  stain  is  generally  used  also. 

S  464.  Counterstaining  with  Eosin. — One  of  the  solutions 
of  eosin  (§  401)  is  dropped  upon  the  sections  after  the  hematoxylin 
has  been  washed  away  with  water.  This  stains  almost  instantly. 
One  rarely  needs  to  stain  with  eosin  over  10  or  30  seconds.  The 
excess  stain  is  then  washed  away  with  a  pipette  or  by  dipping  the 
slide  into  water. 

ij  465.  Dehydrating,  Clearing  and  Mounting. — Puttheslide 
directly  into  95%  alcohol  after  it  is  rinsed  with  water.*  Leave  it 
in  the  alcohol  a  short  time  and  transfer  to  fresh  95  (/fi  alcohol  or  to 
absolute  alcohol  a  few  seconds,  10-20.  One  must  not  leave  the 
sections  too  long  in  the  alcohol  or  the  eosin  will  all  dissolve  out. 

Remove  the  slide  from  the  alcohol  and  put  it  into  a  jar  of  clearer 
(§  392  )  or  put  it  on  the  rack  (Fig.  248,  251)  and  add  enough  clearer 
to  cover  the  sections.  Soon  the  clearer  will  displace  the  alcohol  and 
make  the  sections  translucent.  It  usually  requires  only  half  a 
minute  or  so.  The  clearer  is  drained  off  and  balsam  put  on  the  sec- 
tions, and  then  a  clean  cover-glass  is  added.  One  soon  learns  to 
use  the  right  amount  of  balsam.  It  is  better  to  use  too  much  than 
too  little.  It  is  usually  better  to  press  the  cover  down  very  gently. 
With  some  delicate  objects  like  embryos  in  the  early  stages  this  is 


In  the  past  the  plan  for  changing  sections  from  95%  alcohol  to  water, 
for  example,  has  been  to  run  them  down  gradually,  using  75,  50  and  35% 
alcohol,  successively.  Each  percentage  may  vary,  but  the  principle  of  a  grad- 
ual passing  from  strong  alcohol  to  water  was  advocated.  On  the  other  hand  I 
have  found  that  the  safest  method  is  to  plunge  the  slide  directly  into  water 
from  the  95%  alcohol.  The  diffusion  currents  are  almost  or  quite  avoided  in 
this  way.  There  is  no  time  for  the  alcohol  and  water  to  mix,  the  alcohol  is 
washed  away  almost  instantly  by  the  flood  of  water.  So  in  dehydrating  after 
the  use  of  watery  stains,  the  slide  is  plunged  quickly  into  a  jar  of  95%  alcohol. 
The  diffusion  currents  are  avoided  in  the  same  way,  for  the  water  is  removed 
by  the  flood  of  the  alcohol.  This  plan  has  been  submitted  to  the  severe  test 
of  laboratory  work,  and  has  proved  itself  perfectly  satisfactory  (  1895-1908). 


314 


STAINING  AND  PERMANENT  MOUNTING       [  CH.  X 


not  safe.     A  safe  method  for  all  objects  is  to  add  a  slight  weight, 
and  put  the  slide  in  a  warm  place. 

After  the  balsam  is  quite  dry  the  excess  may  be  scraped  off  the 
slide  with  a  knife  and  then  the  slide  and  cover  cleaned  from  the 
remaining  balsam  by  a  piece  of  gauze  wet  with  xylene.  Finally  the 
slide  should  be  labeled  and  stored. 


FlG.  252.  Coplin's  staining  dish. 
A.  The  entire  dish;  B.  The  dish  in 
cross  section.  This  is  made  of  glass  and 
is  a  very  neat  piece  of  apparatus.  With 
it  ten  slides  may  be  stained  at  once. 
(Cut  loaned  by  the  Whitall  Tatum  Co.) 


CROSS-SECTION 
SHDV/ING  SLIDES 
IN  POSITION. 


§  466.  Counterstaining  with  the  Eosin  in  the  Clearer. — 
With  this  method  the  eosin  is  dissolved  in  the  carbol-xylene  clearer, 
and  the  hematoxylin  stained  sections  are  dehydrated  with  95% 
alcohol  and  absolute  alcohol  if  necessary  and  then  placed  in  the 
clearer.  The  sections  are  cleared  and  stained  in  eosin  at  the  same 
time.  It  usually  takes  half  a  minute  or  more  for  the  double  process. 
When  the  sections  are  clear  and  sufficiently  red,  the  slide  is  removed 
and  the  clearer  drained  off  by  holding  in  the  forceps  or  in  the  drain- 
ing funnel  (Figs.  248,  251).  Then  the  balsam  is  added,  and  cov- 
ered as  described  above. 

It  is  a  good  plan  to  rinse  off  the  stained  clearer  by  pure  xylene 
before  adding  the  balsam.  This  is  not  absolutely  necessary,  how- 
,ever. 

§  467.  Hematoxylin  and  Picro-Fuchsin. — Picro-fuchsin  is 
so  selective  in  its  general  staining  that  it  is  frequently  used  after 
hematoxylin.  The  hematoxylin  staining  should  be  intense  and 
after  the  hematoxylin  is  washed  away  add  the  picro-fuchsin  (§  424). 
It  takes  only  a  few  seconds  for  it  to  act,  10  to  30  seconds.  Wash 
with  distilled  water,  or  natural  water  very  faintly  acidulated.  The 
acid  fuchsin  is  very  sensitive  to  alkalies  and  fades  easily. 


I'll.  X]       STAININC;  AND  PERMANENT  MOUNTING  315 

Dehydrate  in  95  '/c  and  absolute  alcohol,  clear  and  mount  in 
acid  balsam.  Acid  balsam  injures  hematoxylin,  but  is  necessary 
for  the  red  in  the  picro-fuchsin. 

Look  out  for  mercuric  chlorid  crystals  in  the  sections  (§413, 
477)- 

§  468.  Hematoxylin  and  Mucicarmin. — Tissues  and  organs 
are  best  fixed  in  Zenker's  or  mercuric  chlorid.  Small  intestine  is 
one  of  the  most  striking  and  instructive  organs  for  this  double  stain . 
Make  the  sections  by  the  paraffin  method,  but  do  not  fasten  them 
to  the  slide  with  collodion,  for  collodion  stains  with  mucicarmin 

(§  389). 

Stain  i  to  24  hours  in  mucicarmin.  Wash  off  the  stain  with 
water  and  then  stain  with  hematoxylin.  Do  not  stain  too  deeply. 
Wash  with  water,  dehydrate,  clear  and  mount  in  natural  balsam. 
Nuclei  will  be  bluish  or  purple  and  the  cells  containing  mucus  will 
be  rose  red.  The  goblet  cells  of  the  villi  stand  out  like  small  red 
goblets,  and  if  any  mucus  is  streaming  out  of  them  it  will  be  red. 

WEIGERT'S  ELASTIC  STAIN,  WITH  PICRO-FUCHSIN 
AND  MUCICARMIN 

§  469.  Elastic  Stain. — Take  a  slide  of  sections  made  either 
by  the  paraffin  or  the  collodion  method  (§  §  439,  451)  from  alcohol 
and  put  the  slide  into  a  jar  or  a  shell  vial  of  the  stain.  This  is  an 
alcoholic  stain  (§461)  hence  the  sections  should  not  be  washed  in 
water.  Allow  the  stain  to  act  from  ^  hour  to  an  hour.  Wash  off 
the  superfluous  stain  with  95%  alcohol  from  a  pipette  or  by  rinsing 
in  a  jar  of  95  %  alcohol.  It  is  better  in  either  case  to  use  the  pipette 
and  clean  alcohol  for  the  final  washing. 

This  stain  alone  gives  a  bluish  tone  to  the  entire  tissue,  the 
elastic  tissue  being  stained  a  very  deep  blue.  For  greater  contrast 
and  to  bring  out  the  white  fibrous  tissue,  muscle,  etc.,  counter- 
stain  with  picro-fuchsin  of  %  the  strength  given  in  the  regular 
stain  (§  424,  i.  e.,  picro-fuchsin  i  part,  distilled  water  3  parts). 

Dip  the  slide  of  sections  into  distilled  water,  and  then  into  a 
shell  vial  of  the  stain.  Stain  15  to  30  seconds  on  the  average- 
Wash  in  distilled  water  and  dehydrate  in  95%  alcohol  and  absolute 
if  necessary,  then  clear  in  carbol-xylene  and  mount  in  acid  balsam 
(§  387)-  The  elastic  tissue  should  be  almost  black  ;  white  fibrous 


3i6  STAINING  AND  PERMANENT  MOUNTING       [  CH.  X 

tissue  red,  muscle,  blood  and  epithelia  yellow  or  yellowish.     Arter- 
ies are  excellent  for  this  combination. 

§  470.  Combined  Elastic,  Mucicarmin  and  Picro-Fuch- 
sin  Stain. — For  this,  one  should  take  some  object  that  is  known  to 
contain  elastic  tissue,  mucus,  white  fibrous  tissue  and  muscle. 
(The  non-cartilaginous  part  of  the  trachea  is  excellent.)  The  organ 
should  have  been  fixed  in  mercuric  chlorid  or  Zenker's  fluid 
(§  §  416,  429)  for  this  preparation.  The  sections  should  be  made 
by  the  paraffin  method  (§  439)  and  no  collodion  should  be  used  for 
fastening  the  sections  to  the  slide  (§  450)  for  collodion  is  stained  by 
mucicarmin. 

(1)  Stain  first  in  the  elastic  stain   i  hour.     Wash  well  with 
95%  alcohol  and  then  with  water. 

(2)  Stain  in  a  shell  vial  or  jar  of  mucicarmin  (§  389)  from   i 
to  24  hours.     Wash  well  with   water,  but  one  must   be  careful  in 
treating  these  sections  as  they  have  no  collodion  mantle  to  protect 
them. 

(3)  Stain    15  to  30  seconds  with  picro-fuchsin  of   ^  strength 
(§  469).     Dehydrate  with  95%   and  if  necessary  absolute  alcohol. 
Clear  in  carbol-xylene  and  mount   in  acid  balsam   (§   387).     The 
elastic  tissue  will  be  black  or  blue  black.     Mucus  will  be  carmin  or 
rose  red,  white  fibrous  tissue  will  be  magenta  red,  muscle,  epithe- 
lium and  blood  will  be  yellow. 

EOSIN   METHYLENE   BLUE   STAINING 

§471.  Eosin  Methylene  Blue. — One  of  the  best  objects  for 
this  stain  is  a  hemolymph  gland.  Such  a  gland  is  easily  and  surely 
found  by  a  beginner  if  he  takes  the  heart  and  lungs  of  a  veal.  In 
the  fat  around  the  heart  and  behind  the  pleura  will  be  found  red 
bodies  looking  almost  like  blood  clots.  Remove  carefully,  fix  in 
Zenker's  fluid  or  mercuric  chlorid,  (§  §  416,  429).  Section  by  the 
paraffin  method,  make  the  sections  5yu  and  lOyu  thick.  Use  collo- 
dion for  ensuring  the  fixation  to  the  slide  (§  450).  Stain  the 
sections  5  minutes  in  alcoholic  eosin  (§  402).  Wash  off  the  eosin 
stain  with  water.  (This  is  an  exception  to  the  generalization  in 
§  461,  2.) 

Stain  in  methyleue  blue  (§  417)  /^  to  5  minutes.  Rinse  well 
in  tap  water.  Dehydrate  with  neutral  95%  alcohol  (§  380)  and 


CH.X~\  SERIAL  SECTIONING  317 

with  absolute  alcohol.  Work  rapidly  with  only  one  slide  at  once. 
Clear  with  pure  xylene,  mount  in  neutral  balsam  (§  386).  All 
nuclei  should  be  blue  and  all  red  blood  corpuscles,  blight  eosin  red. 
If  one  is  successful  this  is  a  most  striking  and  instructive  prepara- 
tion. Spleen  is  also  very  instructive. 

Eosin-methylene  blue  staining  is  also  excellent  for  demonstrat- 
ing mucus  (§  468). 

Do  not  forget  that  mercury  is  liable  to  be  present  in  sections  of 
tissue  fixed  with  any  mercuric  fixer.  Remove  them  with  iodized 
alcohol  (§  413).  This  should  be  done  before  the  staining.  One 
can  tell  whether  the  tissues  contain  mercury  by  looking  at  the 
unstained  sections.  The  mercury  looks  black  by  transmitted  light, 
white  by  reflected  light.  The  substance  is  often  in  the  form  of 
delicate  black  pins. 

MAKING  SERIES  ;   SERIAL   SECTIONING 

§  472.  General  on  Series. — It  is  coming  to  be  appreciated 
more  and  more  that  in  histology  as  well  as  in  embryology  one  can 
only  get  a  complete  knowledge  of  structure  by  having  the  entire 
organ  cut  in  microscopic  sections  and  each  section  mounted  in  order. 
Furthermore  it  is  necessary  to  have  the  organ  cut  in  three  different 
planes.  In  this  way  one  can  see  every  aspect  of  the  structural  ele- 
ments and  their  arrangement  in  the  organs. 

In  single  sections  one  gets  only  a  partial  view.  For  example, 
how  many  students  have  any  other  idea  of  a  ciliated  cell  than  that 
it  is  a  cell  with  triangular  outline  with  a  brush  of  cilia  at  the  broad 
end.  Probably  many  would  be  puzzled  if  they  had  a  top  view  of 
the  ciliated  end  ;  and  the  attached  end  would  be  even  more 
puzzling. 

It  may  not  be  possible  for  every  worker  to  make  serial  sections 
of  all  the  organs  in  all  the  three  planes,  but  every  one  who  is  work- 
ing seriously  in  histology  can  make  all  his  preparations  serial,  that 
is  the  sections  which  are  mounted  can  be  in  serial  order,  then  a  puz- 
zling appearance  in  one  section  may  be  perfectly  intelligible  in  one 
a  little  farther  along. 

To  get  the  greatest  benefit  from  serial  as  indeed  also  from  single 
sections,  the  sections  should  be  made  in  a  definite  manner,  that  is, 
they  should  be  exactly  across  the  long  axis  of  an  organ  or  parallel 
with  the  long  axis  (Transections,  and  Longisections) . 


SERIAL  SECTIONING 


[C7/.  X 


Or  with  such  an  organ  as  the  liver,  the  skin,  etc.,  the  sections 
may  be  parallel  with  the  surface,  {Surface  Sections^  or  at  right 
angles  to  the  surface  (  Vertical  Sections} . 

ORDER   OF  THE   SECTIONS   IN   A   SERIES 

§  473.  Order  of  Serial  Sections. — Some  plan  must  be 
adopted  in  arranging  the  series  or  only  confusion  will  result.  An 
excellent  plan  is  to  arrange  the  short  pieces  of  ribbons  for  a  given 
slide  as  the  words  on  a  page  are  arranged.  That  is,  section  No.  i 
is  at  the  upper  left  hand  corner.  The  next  row  of  sections  begins 
where  the  first  row  left  off,  etc.,  (Fig.  253). 

As  the  paraffin  stretches  considerably  one  must  cut  the  ribbons 
into  pieces  considerably  shorter  than  the  cover-glass  to  be  used. 


c 
25 


P      I      Q 

V! 
C 

SI      25 


FiG.  253.  Slide  of  an  etnbryologic  series  showing  the  method  of  arranging 
a  sagittal  series.  This  is  the  z$th  slide  of  the  series.  The  sections  are  ar- 
ranged like  the  zvords  and  lines  in  a  book,  i.  e. ,  from  left  to  right.  (From 
" Guide  to  Histology  and  Embryology  in  Cornell  University.") 

Both  the  paraffin  and  collodion  methods  are  adapted  to  the 
preparation  of  series.  The  paraffin  ribbons  are  easier  to  manage 
and  easier  to  make  than  the  serial  sections  in  collodion. 

By  arranging  the  collodion  sections  as  they  are  cut  on  the  knife 
in  collodion  sectioning  (§  457),  one  can  put  them  on  the  slide  in 
perfect  series  by  the  tissue  paper  method  (§  458). 

If  the  sections  are  large,  as  in  cutting  serial  sections  of  the  cen- 
tral nervous  system,  the  series  can  be  kept  in  order  in  a  small  dish 
by  putting  a  piece  of  tissue  paper  over  each  section  and  piling  them 
up.  If  the  vessel  is  small  enough  the  papers  and  sections  will  not 
shift  and  get  out  of  order.  Or  one  might  put  a  single  section  in  a 
Syracuse  watch  glass  and  pile  them  up  in  series  (Fig.  208).  Then 
in  mounting  the  sections  can  be  taken  in  order. 


CH.  X] 


SERIAL  SECTIONING 


§  474.  Numbering  the  Serial  slides. — For  temporary  num- 
bering a  fine  pen  with  Higgins'  waterproof  carbon  ink  serves  well. 
If  the  slide  is  clean  one  can  write  on  it  as  well  as  on  paper.  When 
the  ink  is  dry  it  should  be  coated  with  thin  shellac  or  with  thin 
xylene  balsam.  Sometimes  thin  collodion  is  used.  It  is  also  im- 
portant to  write  the  number  of  the  slide  with  a  writing  diamond. 
The  double  marking  is  desirable  because  with  wet  slides  the  dia- 
mond number  is  hard  to  see,  while  the  ink  marks  are  clearly  visible. 
One  is  not  so  liable  to  wipe  off  the  sections  if  the  ink  mark  is 
present. 


FIG.  254.  Egg  pipette.  This  is  made  by  putting  a  short  piece  of  soft  rubber 
tubing  over  the  end  of  a  glass  pipette  with  rubber  bulb.  With  this  one  can 
handle  the  eggs  both  fresh  and  hardened  without  any  danger  of  injury.  (Jour. 
Appl.  Micr.  1898,  p.  720. ) 

FIG.  255.  Lens  holder.  A 
lens  in  such  a  holder  is  very 
convenient  for  sorting  and 
orienting  small  eggs  or  em- 
bryos in  imbedding.  One  can 
ha  ve  the  eggs  in  a  watch-glass 
of  melted  paraffin  on  a  copper 
warming  plate  (Fig.  241}  and 
arrange  the  eggs  or  embryos 
under  a  lens  in  such  a  lens 
holder.  Then  if  cold  water  is 
poured  on  the  plate  around  the 
-watch-glass  the  paraffin  will 
cool  and  hold  them  in  place. 
(Cut  loaned  by  the  Bausch  &  Lomb  Opt.  Co.) 


FIXING  AND  STAINING    FOR   SERIES 

§  475.  Fixing. — The  two  most  used  fixers  for  embryos  are 
Zenker's  fluid  and  Formaldehyde  (§  406,  429).  For  those  unskilled 
in  microscopic  technic,  or  for  one  who  is  exceedingly  busy  the  best 
results  are  obtained  by  putting  the  embryos  in  formaldehyde,  (10 
parts  of  formalin,  the  formalin  of  the  pharmacy,  and  90  parts  water 
answers  well).  If  there  is  plenty  of  this  the  embryos  are  likely  to 


320  SERIAL  SECTIONS  OF  EMBRYOS  [  CH.  X 

be  well  preserved  even  though  they  are  left  in  the  membranes,   and 
that  is  far  the  best  way  for  small  embryos. 

§  476.  Fastening  the  Sections  to  the  Slide. — For  all  serial 
work  it  is  especially  desirable  to  fasten  the  sections  to  the  slide  with 
collodion  (§  450).  This  should  always  be  done  unless  some  stain 
like  carmin  is  to  be  used  on  the  slide  after  the  sections  are  fastened. 
With  thin  sections,  if  one  is  careful  enough,  an  entire  series  can 
be  carried  through  without  losing  a  section,  but  with  thick  sections 
(i5yM  and  thicker)  some  are  almost  sure  to  separate  from  the  slide. 

§  477.  Removal  of  Mercuric  Chlorid  from  Sections. — It 
should  be  remembered  that  if  a  fixer  containing  mercuric  chlorid  is 
used  the  sections  are  almost  sure  to  contain  mercury.  By  trans- 
mitted light  the  mercury  appears  dark.  Often  the  appearance  is  as 
if  a  multitude  of  delicate  black  pins  were  in  the  section.  Sometimes 
the  mercury  is  in  rounded  masses.  This  should  be  removed  by 
putting  the  slides  of  sections  into  alcoholic  iodin  (§  413).  After 
half  an  hour  or  an  hour  wash  off  the  iodized  alcohol  with  pure  95  '/r 
alcohol  and  the  sections  are  ready  for  staining. 

If  the  embryo  was  stained  in  toto  and  contains  mercury,  the 
sections  should  be  passed  from  the  deparaffining  xylene  to  the 
iodized  alcohol  (§  413).  After  half  an  hour  or  more  the  slides  are 
passed  through  pure  95%  alcohol,  and  back  to  the  xylene  or  to 
carbol-xylene.  Then  they  can  be  mounted  in  balsam. 

§  478.  Staining  for  Series. — There  is  a  great  advantage  in 
point  of  time  and  safety  in  staining  the  entire  embryo  in  some  good 
stain  like  borax  carmin  (§  388).  Carmin  is  a  very  permanent 
stain  also.  For  bringing  out  special  structural  details  the  sections 
are  stained  on  the  slide  as  described  in  §  461-471.  The  slide 
baskets  are  almost  a  necessity  for  serial  work  (Fig.  244,  251),  as  the 
slides  are  handled  individually  only  twice,  ( i )  when  they  are  spread 
and  dried  and  put  into  the  baskets,  and  (2)  after  all  the  processes 
are  complete  and  the  sections  are  to  be  mounted  in  balsam. 

The  sections  are  mounted  in  balsam  directly  from  the  depara- 
ffining xylene.  No  alcohol  is  used  unless  it  is  necessary  to  remove 
crystals  of  mercuric  chlorid  (§  477). 

SERIAL  SECTIONS   OF  EMBRYOS 

§  479.     Serial  Sectioning  Embryos  and  Minute  Animals. — 


CH.  X]  SERIAL  SECTIONS  OF  EMBRYOS  321 

Serial  sections  of  these  should  be  made  in  the  three  cardinal  sectional 
planes,  viz;  Transections;  Frontal  Sections;  Sagittal  Sections. 

If  models  are  to  be  constructed  from  the  sections  it  may  be  more 
conveniently  done  if  the  sections  are  one  of  the  following  thicknesses: 
5ju,  IOJM,  i.5/<,  20/<,  30jw,  4ojw,  50jM,  6o//,  8ojw. 

§480.  Transections,  that  is  sections  across  the  long  axis  of 
the  embryo  or  animal. 

Imbed  the  embryo  with  the  right  side  down,  taking  the  pre- 
caution to  have  a  layer  of  paraffin  between  the  embryo  and  bottom 
of  the  box  (§  441). 

(1)  Mount  the  block  of  paraffin  containing  the  embryo  so  that 
the  tail  end  is  next  the  microtome  holder.     The  head   is  then  cut 
first. 

(2)  Place  in  the  microtome  so  that  the  right  side  of  the  embryo 
meets  the  edge  of  the  knife. 

(3)  Mount  as  a  printed  line  and  the  first  or  cephalic  section  is 
at  the  upper  left  hand  corner,  and  the  dorsal  aspect  of  the  embryo 
is  toward  the  upper  edge  of  the  slide. 

Under  the  microscope  the  rights  and  lefts  appear  as  in  the  observ- 
er's  own  body,  also  the  dorsal  and  ventral  aspects  so  that  he  can 
easily  locate  parts  by  comparing  them  with  his  own  body. 

§481.  Frontal  Sections,  that  is  sections  lengthwise  of  the 
embryo  or  animal  and  from  right  to  left  (dextral  and-  sinistral),  so 
that  the  smbryo  is  divided  into  equal  or  unequal  dorsal  and  ventral 
parts. 

Imbed  the  embryo  with  the  right  side  down  in  the  imbedding 
box  as  before. 

1 i )  Mount  the  paraffin  block  so  that  the  ventral  side  of  the 
embryo  is  next  the  microtome  holder.     The  dorsal  side  is  then  cut 
first. 

(2)  Let  the  right  side  of  the  embryo   meet  the  edge  of  the 
knife. 

(3)  Mount  the  first  section  on   the  left  end  of  the  slides  as 
before  so  that  the  sections  are  crosswise  on  the  slides,  the  tail  toward 
the  upper  edge.     Under  the  compound  microscope  the  head  appears 
toward  the  upper  edge  and  the  rights  and  lefts  are  as  in  the  observer's 
own  body. 

(4)  If  the  sections  are  too  long  to  mount  crosswise  of  the  slide, 
cut  the  sections  apart  and  mount  with  the  head  to  the  right. 


322  SERIAL  SECTIONS  OF  EMBR  YOS  [  CH.  X 

§  482.  Sagittal  Sections,  that  is  sections  lengthwise  of  the 
embryo  or  animal  and  from  the  ventral  to  the  dorsal  side,  thus 
dividing  the  body  into  equal  or  unequal  right  and  left  parts. 

For  these  sections  imbed  the  embryo  with  the  right  side  down 
as  before. 

(1)  Put  the  right  side  of  the  embryo  next  the  microtome 
holder,  then  the  left  side  is  cut  first. 

(2)  L,et  the  caudal  end  meet  the  knife  edge  if  the  embryo  is 
small. 

(3)  Put  the  first  section  in  the   upper  left  hand   part  of  the 
slide  as  in  the  other  cases.     The  sections  will  be  lengthwise  of  the 
slide.     This  brings  the  ventral  side  up  and  the  head  to  the  right  on 
the  slide.     Under  the  microscope  the  head  appears  at  the  left  and 
the  dorsal  side  away  from  the  observer  (Fig.  253). 

(4)  For  large  or  long  embryos  place  the  right   side  next   the 
microtome  holder  as  above,  but  let  either  dorsal  or  ventral  aspect 
meet  the  knife.     Cut  the  sections  apart  and  mount  as  in  (3). 

§  483.  Axes  for  Sections. — For  transections  cut  across  the 
longest  straight  line  from  head  to  tail. 

For  sagittal  sections  select  the  straightest  embryo  and  cut  par- 
allel with  the  longest  axis  dorso- ventral. 

For  frontal  sections  cut  parallel  with  the  long  axis,  dextro- 
sinistral. 

§  484.  For  serial  sections  with  collodion  imbedded  objects  it 
is  a  great  advantage  to  have  the  imbedding  mass  unsymmetrically 
trimmed,  so  that  if  a  section  is  accidentally  turned  over  it  may  be 
easily  noticed  and  rectified. 

Furthermore  it  is  imperatively  necessary  that  the  object  be  so 
imbedded  that  the  cardinal  aspects,  dextral  and  sinistral,  dorsal  and 
ventral,  cephalic  and  caudal,  shall  be  known  with  certainty. 

§  485.  Thickness  of  Cover-Glass  for  Serial  Sections.— 
It  is  a  great  advantage  to  use  very  thin  cover-glasses  (0.12-0.18 
mm.)  for  serial  sections,  then  the  cover  will  not  prevent  the  use  of  high 
powers.  When  the  ordinary  slides  (25X76  mm.,  1X3  inch)  are 
used,  cover-glasses  24 X  50  mm.  may  be  advantageously  employed. 

The  combined  thickness  of  the  sections  on  a  slide  is  easily 
determined  by  multiplying  the  number  of  sections  by  the  thickness 
of  each. 


CIL  .V] 


SERIAL  SECTIONS  OF  EMBRYOS 


323 


S  486.  Labeling  Serial  Sections. — The  label  of  a  slide  on 
which  serial  sections  are  mounted  should  contain  at  least  the 
following  : 

'The  name  of  the  embryo  and  the  number  of  the  series  ;  the 
number  of  the  slide  of  that  series  ;  the  thickness  of  the  sections, 
and  the  number  of  the  first  and  last  section  on  the  slide  ;  the  date. 
It  is  also  a  convenience  to  have  the  information  repeated  in  part  on 
the  left  end. 


FIG.  256.  Removable  mechanical  stage.  It  fits  any  square  stage  and  has 
the  advantage  of  large  motion  in  both  directions  making  it  especially  useful 
for  the  study  of  serial  sections.  ( Cut  loaned  by  the  Spencer  Lens  Co. ) 

REFERENCES 

For  sectioning  staining,  etc.,  in  the  various  ways  see  :  Lee,  Mann, 
Ehrlich,  Mallory  and  Wright.  The  Microscopic  Journals. 

For  the  preparation  of  Embryos  see  Foster  and  Balfour's  Elements  of 
Embryology.  Minot's  Laboratory  Text-Book  of  Embryology.  Consult  also 
the  general  Bibliography  at  the  end. 


324  DRAWINGS  FOR  BOOK  ILLUSTRATION  \_CH.X 

DRAWINGS    FOR    PUBLICATION 

§  487.  Preparation  of  Drawings. — The  inexpensive  processes  of  reproduc- 
ing drawings  bring  within  the  reach  of  "every  writer  upon  scientific  subjects 
the  possibility  of  presenting  to  the  eye  by  diagrams  and  drawings  the  facts  dis- 
cussed in  the  text.  Though  artistic  ability  is  necessary  for  perfect  representa- 
tion of  an  object,  neatness  and  care  will  enable  anyone  to  make  a  simple  illus- 
trative drawing,  from  which  an  exact  copy  can  be  obtained  and  a  plate  pre- 
pared for  printing. 

A  careful  study  of  the  cuts  or  plates  used  to  illustrate  the  same  class  of 
facts  as  one  wishes  to  show  will  enable  one  to  produce  similar  effects.  Out- 
lines which  are  transferred  to  the  drawing  paper  may  be  obtained  by  the 
camera  lucida,  the  projection  microscope  (Figs.  257-258),  or  from  a  photo- 
graph. The  drawing  should  be  made  so  that  it  can  be  reduced  anywhere 
from  one-eighth  to  one-half.  For  ordinary  photo-engraving  for  such  line 
drawings  as  are  used  to  illustrate  this  book,  use  perfectly  black  carbon  ink.  A 
shaded  or  wash  drawing  can  be  reproduced  by  the  half-tone  process,  also  photo- 
graphs as  is  illustrated  by  figures  79,  82,  89-92,  180-182.  A  crayon  drawing  on 
stipple  paper  with  shadows  re-enforced  by  ink  lines  and  high  lights  scratched 
out  with  a  sharp  knife  give  admirable  results  for  anatomic  figures  by  the  half- 
tone process.  For  examples  see  the  various  volumes  of  the  American  Journal 
of  Anatomy.  In  vol.  iv.  pp.  409-443,  and  in  vol.  viii,  pp.  17-47,  one  will  find 
in  the  accompanying  plates  pure  line  drawings',  half  tones  from  photographs, 
and  half  tones  from  shaded  drawings. 

§  488.  The  Lettering  on  Drawings. — For  half-tones  this  should  be  done 
directly  on  the  drawing,  as  illustrated  by  the  plates  just  referred  to. 

For  photographic  reproduction  of  line  work,  letters,  numerals  or  words 
used  to  designate  the  different  parts  can  be  put  on  the  drawings  by  pasting 
the  printed  letters  etc.  of  the  proper  size  in  the  right  position.  In  preparing 
the  block  the  engraver  removes  all  shadows  from  the  edge  so  that  the  letters 
look  as  if  printed  on  the  drawing.  If  tissue  paper  were  used  on  which  to  print 
the  letters  the  engraver  would  have  less  trouble  in  removing  shadows  around 
the  edge  of  the  paper. 

Letters  and  figures  should  be  distinct,  but  not  so  large  that  they  are  the 
most  conspicuous  feature  of  the  drawing. 

MODELS    FROM    SERIAL   SECTIONS 

\  489.  General  Considerations  on  Modeling. — Anatomists  have  for  a  long 
time  produced  models  of  gross  anatomic  specimens,  and  enlarged  models  for 
minute  details. 

Naturally  after  serial  sections  of  embryos  and  organs  came  to  be  made 
with  considerable  accuracy  and  of  known  thickness,  there  was  a  desire  to 
make  enlarged  models  which  should  be  exact  representations  of  the  original 
rather  than  the  generalized  approximations  built  up  as  an  artist  produces  a 
statue. 


CH.  X}  WAX  MODELS  325 

Further  the  difficulty  of  getting  a  true  conception  of  the  object  by  study- 
ing only  two  dimensions  in  the  sections  is  very  great,  hence  a  model  giving 
all  three  dimensions  becomes  almost  a  necessity  for  the  beginner  in  embry- 
ology, and  is  of  enormous  advantage  to  an  investigator  in  working  out  the 
true  form  and  relation  of  complex  structures. 

The  principles  involved  in  the  construction  of  a  model  are  exceedingly 
simple  : — 

1.  It  is  necessary  that  the  embryo  or  other  object  to  be  modeled  should 
be  cut  into  a  series  of  sections  of  definite  thickness. 

2.  The  sheets  of  modeling  material  must  be  as  much  thicker  than  the 
sections  as  the  model  is  to  be  larger  than  the  original. 

3.  The  sections  must  be  drawn  as  much  larger  than  the  actual  specimen 
as  the  model  is  to  be  larger  than  the  object. 

4.  The  drawings  with  the  desired  outlines  must   be   made   directly  upon 
or  transferred  to  the  sheets  of  modeling  material  which  are  then  cut  out,  fol- 
lowing the  lines  of  the  drawing. 

5.  The  different  plates  of  modeling  material  representing  all  the  sections 
are  then  piled  up,  in  order,  thus  giving  an  enlarged  model  of  the  object  with 
all  its  parts  in  proper  position  and  in  true  proportions. 

MODELS    OF    WAX 

\  490.  Wax  Models. — For  making  wax  models,  bees-wax  820  grams, 
paraffin  270  grams,  and  resin  25  grams,  are  melted  together  and  thoroughly 
mixed. 

To  get  the  sheets  of  wax  of  the  proper  thickness  two  methods  are 
available  : — 

The  hot  wax  is  poured  into  a  vessel  containing  hot  water.  The  wax 
spreads  out  into  an  even  layer  over  the  hot  water  and  is  allowed  to  cool. 
While  it  is  solidifying  it  should  be  cut  free  from  the  edges  of  the  vessel.  Of 
course  by  calculation  and  experiment  one  can  put  in  the  right  amount  of  wax 
to  get  a  plate  of  a  given  thickness. 

( 2)  One  must  have  a  wax-plate  machine  consisting  of  a  flat  surface — 
planed  cast  iron  is  good — with  some  means  of  obtaining  raised  edges.  If  these 
are  adjustable  by  a  micrometer  screw  it  is  simple  to  set  them  properly  for  the 
desired  thickness  of  plate.  Then  there  must  be  a  hot  roller.  The  hot  wax  is 
poured  on  the  plate  and  with  the  hot  roller  resting  on  the  raised  edges,  the 
wax  is  rolled  out  into  a  plate.  It  cools  quickly  and  may  be  removed  for 
another  plate.  This  is  the  most  rapid  and  satisfactory  method  of  prepar 
ing  the  plates.  By  using  a  brush  with  turpentine  the  paper  with  the  drawing 
can  be  wet  and  then  with  the  hot  roller  cemented  to  the  plate  before  that  has 
been  removed  from  the  machine. 

The  wax  plate  is  cut  with  a  sharp  instrument,  following  the  outlines  of 
the  object  which  has  been  traced  upon  it  by  the  aid  of  a  camera  lucida  or  the 
projection  microscope.  The  sections  are  piled  together,  some  line  or  lines 
obtained  from  a  drawing  or  photograph  of  the  specimen  before  it  was  imbedded 


326  BLOTTING  PAPER  MODELS  [CI1.X 

and  sectioned  being  used  as  a  guide  by  which  the  correct  form  of  the  pile  of 
sections  can  be  tested.  Finally  the  whole  is  welded  into  one  by  the  use  of 
hot  wax  or  a  hot  instrument.  Models  which  illustrate  complex  internal  struc- 
tures are  difficult  to  prepare,  but  numerous  devices  will  occur  to  the  worker,  as 
the  representation  of  blood  vessels  and  nerves  by  strings  or  wires.  A  large 
model  will  need  much  support  which  can  be  given  by  wire  gauze,  wires,  pins 
or  paper  according  to  the  special  needs. 

A  practical  method  for  wax  modeling  was  first  published  by  G.  Born,  Arch, 
f.  Mikr.  Anat. ,  Bd.  xxii,  1883,  p.  584.  The  most  detailed  statements  of  im- 
provements of  the  method  have  been  published  by  Born  (Bohm  u.  Oppel) 
1904,  and  by  Dr.  F.  P.  Mall  and  his  assistants.  See  contributions  to  the 
Science  of  Medicine,  pp.  926-1045.  Proceedings  of  the  Amer.  Assoc  Anatom- 
ists, 1901,  I4th  session  (1900)  p.  193.  A.  G.  Pohlman,  Zeit.  wiss  Mikroskopie, 
Bd.  xxiii,  1906,  p.  41. 

To  overcome  the  difficulty  of  cutting  outthe  wax  plates,  Dr.  E.  L.  Mark 
of  Harvard  University  uses  an  electrically  heated  wire  moved  rapidly  by  a 
modified  sewing  machine  (Amer.  Acad.  Arts  and  Sciences,  March,  1907; 
Science,  vol.  xxv,  1907  ;  Anat.  Record  April,  1907. 

MODELS   OF   BLOTTING    PAPER 

§  491.  Comparison  of  Wax  and  Paper  Models. — Wax  has 
certain  inherent  defects  for  models  :  It  is  expensive,  heavy  and 
fragile.  It  is  easily  deformed  by  the  temperature  of  summer,  and 
the  amount  of  time  necessary  for  the  preparation  of  the  plates  is 
great.  A  wax-plate  machine  is  expensive  and  bulky. 

It  therefore  seemed  worth  while  to  see  if  there  was  not  some 
other  material  obtainable  in  the  open  market  which  would  be  more 
suitable  and  more  generally  available. 

Blotting  paper  seemed  promising,  and  an  actual  trial  showed  it 
to  be  admirably  adapted  for  the  purpose.  Since  making  the  first 
model  in  1905  it  has  been  constantly  used  in  the  laboratory  of 
embryology  in  Cornell  University.  Models  made  from  it  were 
demonstrated  before  the  Association  of  American  Anatomists  in 
1905  and  before  the  International  Congress  of  Zoology  in  1907. 

"  The  advantages  of  blotting  paper  models  are  the  ease  and 
cleanliness  of  their  production  and  the  lightness  and  durability  of 
the  product.  The  models  are  broken  with  difficulty,  are  easily 
packed  or  transported,  and  when  they  cleave  apart  are  easily 
repaired,  thus  contrasting  with  the  weight  and  fragility  of  wax 
models  and  their  deformation  by  heat.  " 

"  By  this  process  are  secured  for  the  original  model  recon- 
structed from  microscopic  sections,  the  same  qualities  which  have 


Cff.    X]  PLOTTING  PAPER  MODELS  327 

made  the  Auzoux  models  molded  from  papier-mache  such  useful  and 
lasting  additions  to  laboratory  equipment  ;  and  in  the  hands  of  Dr. 
Dwight  and  Mr.  Emerton,  of  Harvard  University,  have  aided  so 
much  in  the  demonstration  of  structure  and  form  of  special  anatomic 
preparations.  " 

£  492.  Thickness  of  Blotting  Paper. — Blotting  paper  of  a 
uniform  thickness  of  i  mm.  f^mm.  and  yz  mm.  were  found  in  the 
market.  The  i  mm.  is  known  as  140  Ib.  A.  and  costs  about  two 
cents  for  a  sheet  61  X  48  centimeters  (24X  19  in.).* 

The  thickness  is  easily  tested  by  cutting  out  50  small  pieces, 
piling  them,  dipping  one  end  in  melted  paraffin  and  pressing  them 
together.  The  whole  pile  should  of  course  measure  50  mm.  if  the 
paper  is  millimeter  paper. 

£  493.  Size  of  the  Model. — In  deciding  upon  the  size  of  the 
model  to  be  made  from  a  given  series  of  sections  one  should  select 
the  largest  section  and  with  the  projection  microscope  throw  the 
image  on  the  table  (Fig.  258).  By  using  different  objectives  and 
different  distances  from  the  microscope  one  can  find  a  size  which 
seems  suitable.  The  magnification  may  be  found  by  §  207.  Then 
by  multiplying  the  whole  number  of  sections  by  the  thickness  of 
the  sections  and  this  by  the  magnification  one  can  get  the  length  or 
height  of  the  model.  One  must  take  these  preliminary  steps  and 
decide  upon  the  magnification  to  be  used  or  the  model  is  liable  to 
be  too  large  to  be  manageable  or  too  small  to  show  well  the  neces- 
sary detail. 

(i)  Suppose  the  model  is  to  be  100  times  the  size  of  the 
original  object,  and  the  object  has  been  cut  into  a  series  of  sections 
io/<  thick.  Then  each  section  must  be  represented  by  a  plate  or 
sheet  100  times  as  long,  broad  and  thick  as  the  object.  As  the 
sheets  of  blotting  paper  are  so  large  (61X48  cm.)  one  need  be 
solicitous  only  about  the  thickness. 

As  each  section  is  actually  io//  thick  and  the  model  is  to  be  100 
times  enlarged,  the  thickness  representing  each  section  must  be 


*Book -stores,  paper  dealers  and  job  printers  are  supplied  by  the  paper 
manufacturers  with  samples  of  blotting  paper.  One  can  look  these  samples 
over,  select  and  order  the  kinds  desired.  The  millimeter  blotting  paper 
mentioned  in  the  text  is  one  of  the  cheaper  grades,  costing  by  the  package  of 
500  sheets  about  two  cents  a  sheet  (sheets  61  X48  centimeters,  24x19  inches). 


328  BLOTTING  PAPER  MODELS  [  CH.  X 

io/<X  IOO=IOOO,M  or  i  millimeter,  i  millimeter  blotting  paper  is 
used  and  every  section  of  the  series  is  drawn. 

(2)  If  the  blotting  paper  were  only  y9^  mm.  thick  it  would  be 
simpler  to  make  the  model  90  times  the  size  of  the  original.  If, 
however,  one  wished  the  magnification  to  be  100,  it  could  be 
accomplished  thus  :  Each  section  in  the  series  should  be  repre- 
sented by  i  mm.  or  iooo/*  in  thickness.  But  if  one  uses  blotting 
paper  of  f^  mm.  thickness  or  goo/u,  there  is  a  loss  of  zooyw  for  each 
section  and  for  9  sections  there  would  be  a  loss  of  goo/*  or  the 
thickness  of  a  sheet  of  the  blotting  paper.  To  remedy  this  one 
uses  10  sheets  of  blotting  paper  for  9  sections.  This  keeps  the 
model  in  true  proportion.  In  practice  each  of  the  sections  is  drawn 
upon  one  sheet  except  one  of  them  and  for  that  two  sheets  of  the 
blotting  paper  are  united  and  the  sections  drawn  upon  the  double 
sheet. 

§494.  General  Rule  for  the  Use  of  Blotting  Paper.— 
Divide  the  thickness  by  which  each  section  is  to  be  represented  in  the 
model  by  the  thickness  of  one  sheet  of  the  blotting  paper  available. 
The  quotient  shows  the  number  of  sheets  or  the  fraction  of  a  sheet 
required  for  each  section. 

If  a  quotient  is  a  mixed  number  reduce  it  to  a  fraction.  The 
numerator  represents  the  number  of  sheets  required  and  the  denom- 
inator the  number  of  sections  to  go  with  the  sheets. 

Examples :  (a)  With  a  series  of  10/1  sections  to  be  modeled 
at  100  enlargement  each  section  of  the  series  must  be  represented  in 
the  model  by  a  thickness  of  io/*X  ioo=iooo/<  or  i  millimeter.  If 
one  uses  millimeter  or  looo/u  paper  then  iooo/t-7-iooo/<=T,  and  one 
must  use  i  sheet  for  i  section. 

(b)  With  a  series  of  10/1  sections  to  be  made  into  a  model   100 
times  enlarged,  and  with  blotting  paper  of  T97  mm.  or  900/1  thick- 
,ness,  each  section  must  be  represented  by  iojwX  100=1000^1.     If  the 

blotting  paper  is  goo/*  thick,  then  it  requires  for  each  section  : 
1000-1-900=1^  sheets  of  paper  or  y  sheets  for  one  section  or  10 
sheets  for  9  sections,  that  is  a  double  sheet  for  one  of  the  nine 
sections. 

(c)  With  a  series  cut  i5/<,  for  a  50  fold  model.     Each  section  is 
represented  by  a  thickness  of  1 5/<X  50=750/4.     If  one  uses    r    mm. 
or  loco/*  blotting  paper  then  each  section  requires  j  50 -^- 


CH.  A'] 


DRAWINGS  FOR  MODELS 


329 


of  a  sheet  for  one  or  3  sheets  for  4  sections.  In  this  case  one  omits 
every  fourth  section  in  drawing,  thus:  ist,  2d,  and  3d  sections 
would  be  drawn  ;  then  the  5th,  6th  and  yth  ;  gth,  roth,  nth,  etc., 
every  fourth  being  omitted. 

(d)  If  for  the  model  just  considered  one  had  T97  mm.  or  900;* 
paper  then  750-1-900=  |.  That  is  there  must  be  5  sheets  of  the 
paper  for  each  6  sections.  In  that  case  every  sixth  section  would 
be  omitted  in  the  drawing  as  every  fourth  section  was  omitted  in  (c). 


FIG.  257.  Abbe  Camera  Lucida  in  connection  with  Bernhard's  drawing 
board.  The  drawing  board  is  adjustable  vertically  for  a  greater  or  less  image 
distance.  It  may  also  be  elevated  toward  the  microscope  to  prevent  distortion 
(Fig.  129}.  The  base  board  is  hinged  so  that  microscope  and  board  may  be 
inclined  together  (Zeiss'  Catalog). 

It  is  of  course  best  to  use  sheets  of  exactly  the  right  thickness 
to  represent  the  necessary  thickness  in  the  model,  (a)  but  one  can 
produce  models  with  accuracy  by  duplicating  one  or  more  sheets 
for  a  group  of  sections  (b)  or  by  omitting  certain  sections  of  the 
series  in  drawing  (c,  d). 

§  495.  Drawings  for  Models. — For  drawing  one  may  use 
the  camera  lucida  (Figs.  128,  132,  257),  taking  the  precautions  to 


330 


DRA  WINGS  FOR  MODELS 


[  CH.  X 


avoid  distortion  (§  204).  For  getting  the  exact  magnification 
desired  one  has  recourse  to  different  oculars,  objectives  and  distance 
of  the  drawing  surfaces  (§  177,  206  E). 

By  far  the  most  satisfactory  means  for  making  the  numerous 
drawings  of  all  sizes  of  object  and  all  magnifications  except  the  high- 
est, is  the  projection  microscope  (  Fig.  258). 

One  can  draw  directly  upon  blotting  paper,  but  it  is  so  import- 
ant to  have  a  drawing  to  refer  back  to  that  one  or  more  duplicates 
should  be  made.  This  is  easily  accomplished  by  putting  a  sheet  of 
carbon  manifolding  paper  on  the  blotting  paper  and  a  sheet  of  thin 


FIG.  258.  Room  and  Apparatus  for  Drawing  with  the  Projection  Micro- 
scope. R.  Radiant,  an  arc  lamp  with  carbons  at  right  angles;  L.  t.  Lamp 
and  microscope  table;  C.  Condenser  with  W.  a  large  water  bath  between  the 
lenses  to  absorb  the  heat  rays.  S.  w.  Stage  and  stage  water  bath  on  which 
rests  the  object  and  keeps  the  object  cool  by  radiation  as  well  as  by  absorption; 
O.  The  objective  representing  the  microscope;  M.  M^irror  at  45°  on  a  draw- 
ing table,  (Dt.)  As  the  microscope  is  horizontal  so  that  the  axial  ray  is  re- 
flected downward  at  right  angles  by  the  45°  mirror  there  is  no  distortion. 
The  scale  of  the  drawing  is  added  exactly  as  described  in  \  207. 


C/l.  -Y]  PREPARATION  OF  MODELS  331 

paper  over  the  carbon  paper  using  thumb  tacks  to  hold  the  blotting 
paper  and  the  duplicating  sheets  in  position. 

One  should  take  the  precaution  to  number  each  drawing  as  it 
is  made  then  confusion  in  the  later  processes  will  be  avoided. 

S  496.  Cutting  out  the  Sheets  for  the  Model.  — "  With  the 
blotting  paper,  if  the  drawings  are  small  the  cutting  is  easily  done 
with  scissors  or  a  knife.  When  the  drawings  are  large  and  espec- 
ially when  the  model  is  to  be  made  by  representing  each  section  by 
two  or  more  thicknesses  of  blotting  paper  it  has  been  found  that  an 
ordinary  sewing-machine  can  be  used  to  do  the  cutting.  By  setting 
the  regulator  for  the  shortest  stitch  an  almost  continuous  cut  is 
made  and  the  parts  are  easily  separated.  If  a  large  sewing-machine 
needle  is  sharpened  in  the  form  of  a  chisel,  the  cut  becomes  consid- 
erably smoother.  It  has  been  found  advantageous  when  long  con- 
tinued or  heavy  work  is  to  be  done  to  attach  to  the  machine  an 
electric  sewing-machine  motor.  Skill  in  guiding  the  work  is  soon 
acquired.  There  are  some  details  of  a  complicated  drawing  which 
are  more  easily  cut  by  the  scissors  or  a  knife  after  the  main  lines 
have  been  cut  by  the  machine." 

§  497.  Contrasting  Colors  for  Marking  Groups  of  Sec- 
tions.— "It  is  a  great  advantage  in  any  working  model  to  have  sec- 
tions at  regular  intervals  in  marked  contrast  with  the  body  of  the 
material.  Blotting  paper  of  a  large  variety  of  colors  (black,  red 
blue,  pink)  is  easily  obtained  in  the  market.  In  the  models  made 
every  tenth  plate  was  a  bright  or  light  color  and  every  icoth  was 
black,  rendering  rapid  numeration  easy." 

S  498.  Putting  the  Sheets  together  to  Make  the  Model. 
"  When  the  paper  sections  are  thus  prepared  they  are  piled  and 
replied  as  is  usual  until  the  shape  conforms  to  an  outline  predeter- 
mined from  photographs,  drawings,  or  measurements  made  before 
the  specimen  was  cut." 

' '  It  has  been  found  that  an  easily  prepared  support  and  guide 
for  the  model  in  process  of  setting  up,  is  made  by  cutting  the  out- 
line to  be  followed  from  a  block  of  four  or  five  sheets  of  blotting 
paper,  marking  upon  it  the  lines  of  direction  of  every  tenth  or 
twentieth  section.  The  colored  numerating  plates  must  of  course 
conform  to  the  spacing  and  direction  of  these  lines." 

"The   preliminary   shaping  having   been  accomplished   more 


332  PREPARATION  OF  MODELS  [CH.X 

| 

exact  modeling  is  undertaken.  The  paper  sections  slide  very  easily 
upon  one  another.  The  most  satisfactory  means  of  fastening  them 
together  is  by  the  use  of  ribbon  pins,  ordinary  pins,  or  wire  nails  of 
various  sizes,  depending  on  the  size  of  the  model.  No  kind  of  paste 
or  glue  was  found  suitable  for  this  purpose." 

§  499.  Finishing  the  Model. — "When  the  model  is  well 
formed,  inequalities  are  best  removed  by  rubbing  with  the  edge  of 
a  dull  knife  and  smoothing  with  sand  paper.  Any  dissections  of 
the  model  for  showing  internal  structures  should  be  planned  for  at 
this  stage  for  it  is  now  more  easily  separated  than  later.  It  is  also 
at  this  time  that  superfluous  "bridges,"  which  have  been  left  in 
place  to  support  detached  parts,  would  better  be  removed." 

"To  finish  the  model  it  is  held  together  firmly  and  coated  with 
hot  paraffin  either  by  a  camels  hair  brush  or  by  dipping  in  paraffin 
and  removing  the  superfluous  coating  by  a  hot  instrument.  One 
might  use  a  thermo-cautery  for  this  purpose." 

"The  paraffin  renders  the  model  almost  of  the  toughness  ot 
wood  without  destroying  the  lightness  of  the  paper." 

§  500.  Coloring  the  Surface;  Dissectng  the  Model.— 
"For  coloring  the  surface  of  the  model,  it  was  found  most  desirable 
to  use  Japanese  bibulous  paper,  lens  paper  (§  125)  which  had  been 
dipped  in  water  color  and  dried.  Any  of  the  laboratory  dyes  or  inks 
can  be  used,  such  as  eosin,  picric  acid,  methylene  green,  black  ink, 
etc.  The  colored  lens  paper  molds  over  the  surface  with  ease  and 
is  held  in  place  by  painting  with  hot  paraffin.  All  color  and  enum- 
eration lines  and  fine  modeling  show  through  the  transparent  paper." 
"When  the  model  ceases  to  be  a  working  model  it  can  be  cov- 
ered with  oil  paints  mixed  with  hot  paraffin  and  rubbed  to  any 
degree  of  finish  desired." 

"One  can  dissect  a  model  by  a  hot  knife  run  along  the  planes 
of  cleavage  or  cut  across  them  by  a  saw. ' ' 

For  the  literature  of  blotting  paper  models  see  :  Susanna  Phelps  Gage, 
Amer.  Jour.  Anat.,  vol.  v,  1906,  p.  xxm  ;  Proceedings  of  the  International 
Zoological  Congress  for  1907;  Anatomical  Record,  Nov.  1907.  (From  this 
paper  the  above  quotations  were  made).  Zeit.  wiss.  Mikroskopie.  Bd.  xxv., 
1908,  pp,  73-75. 

"  Blotting  paper  models  have  also  been  made  and  demonstrated  by  Dr.  J.  H. 
Hathaway  and  by  Dr.  J.  B.  Johnston  at  the  Association  of  American  Ana- 
tomists held  in  New  York,  1906  (Proc.  Assoc.  Amer.  Anatomists,  Anat.  Record 
April  i,  1907). 


BIBLIOGRAPHY 


The  books  and  periodicals  named  below  in  alphabetical  order  pertain  wholly  or  in  part 
to  the  microscope  or  microscopical  methods.  They  are  referred  to  in  the  text  by  recogniza- 
ble abbreviations. 

For  current  microscopical  and  histological  literature,  the  Journal  of  the  Royal  Micro- 
scopical Society,  the  Zoologischer  Anzeiger,  and  the  7-eitscrift  ftlr  wissenschaftliche  Mikros- 
kopie,  Anatomischer  Anzeiger,  Biologisches  Centralblatt  and  Physiologisches  Centralblatt, 
the  Journal  of  Applied  Microscopy  and  Laboratory  methods  and  the  smaller  microscopical 
journals  taken  together  furnish  nearly  a  complete  record.  See  also  the  list  of  periodicals. 

References  to  books  and  papers  published  in  the  past  may  be  found  in  the  periodicals  just 

named,  in  the  Index  Catalog  of  the  Surgeon  General's  library,  in  the  Royal  Society's  Catalog' 

nt i tic  I'tif'frs,  and  in  the  bibliographical  references  given  in  special  papers.     A  full  list 

of  periodicals  may  also  be  found  in  Vol.  XVI  of  the  Index  Catalog,  and  in  later  volumes,  the 

new  ones  are  given. 

BOOKS 

Abbe,  E. — Gesammelte  Abhandlungen.  Bd.  II.  Pp.  346,  Illust.  Jena,  1906.  This  volume 
deals  with  the  microscope,  etc.  Edited  by  Dr.  E.  Wandersleb. 

Adams,  G. — Micrographia  illustrata,  or  the  microscope  explained,  etc.  Illust.  4th  ed., 
London,  1771. 

Adams,  George. — Essays  on  the  Microscope.    4  to.    Illust.    London,  1787. 

AngstrOm.— Recherches  sur  le  spectre  solaire,  spectre  normal  du  soleil.     Upsala,  1868. 

Anthony,  Wm.  A.,  and  Bracket!,  C.  F. — Elementary  text-book  of  physics.  6th  ed.  Pp. 
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JUBL1OGRAPIIY  335 

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Vogel,  H.  W.— Practische  Spectralanalyse  irdischer  stoffe  :  Anleitung  zur  Benutzung  der 
Spectralapparate  in  der  qualitativen  und  quantitative!!  chemische  Analyse  organischer  and 
unorganscher  Korper.  2d  ed.  Figs.  Berlin,  1889. 

Wall,  O.  A.— Notes  on  Pharmacognosy.     2d  ed      Pp.  703.     Illust.     St.  Louis,  1902. 

Walmsley,  W.  H.— The  A,  B,  C  of  Photo-Micrography.  A  practical  hand-book  for 
beginners.  Plates  and  text  figures.  New  York,  1902; 

Weinschenk,  E. — Anleitung  zum  Gebrauch  des  Polarisationsmikroskopes.  Pp.  147. 
Illust.  Freiburg,  1906.  .iluv 

Wethered,  M.— Medical  microsco:  .  Figs.     London  and  Philadelphia.  iS:.2. 

Whipple,  G.  C. — The  Microscopy  of  Drinking  Water,  ad  ed.  Pp.  xii  -  303.  Illust.  New 
York,  1905. 

White,  T.  C. — The  Microscope  and  how  to  use  it.  A  hand-book  for  beginners,  with  chap- 
ters on  marine  aquaria  and  the  staining  of  bacteria.  Illust.  London,  1907. 

Whitman,  C.  O.— Methods  of  research  in  microscopical  anatomy  and  embryology.  Pp. 
255.  Illust.  Boston,  1885. 

Whittaker,  E   T. — The  theory  of  optical  instruments.     Pp.  72.     Cambridge,  1907. 

Wilder  and  Gage  — Anatomical  technology  as  applied  to  the  domestic  cat.  An  introduc- 
tion to  human,  veterinary  and  comparative  anatomy.  Pp.  575,  130  Figs.  2d  ed.  New  York 
and  Chicago,  1886. 

Wiley,  Harvey  W. — Foods  and  their  Adulterations.  The  origin,  manufacture  and  com- 
position of  food  products.  Description  of  common  adulterations,  food  standards  and  national 
food  laws  and  regulations.  Pp.  625.  Illust.  Philadelphia,  1907. 

Wilkinson,   F.— The  study  of  the  Cotton  Plant.     New  York,  1899. 

Williams,  H.  V.— Bacteriology.  4th  edition  revised  and  enlarged  by  R.  Meacle  Bolton. 
PP-  357-  Illust.  Philadelphia,  1003. 

Wilson,  C.  E.  A.— Elements  of  Applied  Microscopy.  Pp.  168.  Illust.  New  York  and 
London,  1905. 

Wilson,  Edmund  B.,  with  the  citoperation  of  Edward  Learning.— An  atlas  of  fertilization 
and  karyokinesis.  Columbia  University  Press,  New  York.  1895.  This  atlas  marks  an  era  in 
embryological  study.  It  has  admirable  text  and  diagrams,  but  the  distinguishing  feature  is. 
the  large  number  of  almost  perfect  photo-micrographs. 

Winkelmann,  A. — Handbuch  der  Physik,  2  Aufl.  Bd.  vi,  I  Optik.  Pp.  432.  Illust. 
Leipzig,  1904. 

Winslow,  Charles-Edward  Amory. — Elements  of  applied  Microscopy.  A  text-book  for 
beginners.  Pp.  183.  Illust.  New  York,  1905. 


IUI1LIOGRAPHY  343 

Winton,  Andrew  I,.,  in  Collaboration  with  Dr.  J.  Moeller. — The  Microscopy  of  Vegetable 
Pp.  701.  Illust.  New  York,  1906. 

\Vond,  J.  ("..—Common  objects  for  the  microscope.  I'p.  132.  London,  no  date.  Upwards 
of  400  figures  of  pretty  objects  for  the  microscope,  also  brief  descriptions  and  directions  for 
preparation. 

Worinly,  T.  G. — The  micro-chemistry  of  poisons.     2d  ed.     Pp.  742.     Illust.     Phila.,  1885. 

Wright,  Sir  A.  K.— Principles  of  Microscopy,  being  a  hand-book  to  the  Microscope.  Pp. 
250.  Illust.  New  York,  1907. 

Wright,  Lewis.— A  popular  hand-book  to  the  Microscope.     Pp.  256.     Illust.    London,  1885. 

Wright,  Lewis.— Optical  Projection,  a  treatise  on  the  use  of  the  lantern  in  exhibition  and 
scientific  demonstration.  4th  ed.  Pp.  450.  Illust.  London,  1906.  (Beginners  will  find  this 
book  very  helpful.) 

Wythe,  J.  H. — The  niicroscopist  ;  a  manual  of  microscopy  and  a  compendium  of  micro- 
scopical science.  4th  ed.  Pp.  434,  252  Figs.  Philadelphia,  1880. 

/immermann.  Dr.  A.— Das  Mikroskop.  ein  Leitfadeii  der  wissenschaftlichen  Mikro- 
skopie.  Illust.  Leipzig  und  Wein,  1895. 

See  also  Watt's  chemical  dictionary,  and  the  various  general  and  technical  encyclopedias. 

I'KKIODICALS* 

The  American  Journal  of  Anatomy,  Baltimore,  1901  -f.  The  American  Journal  of  Anat- 
omy including  Histology,  Embryology  and  Cytology  was  established  by  seven  universities,— 
Harvard,  Johns  Hopkins.  Columbia,  Pennsylvania,  Michigan,  Cornell  and  Chicago. 

It  is  now  (1908),  published  under  the  auspices  of  the  Wistar  Institute  of  Anatomy  and 
Biology,  Philadelphia,  and  lias  an  editorial  board  consisting  of  Charles  R.  Bardeeu,  Univer- 
sity of  Wisconsin  ;  Henry  H.  Donaldson,  the  Wistar  Institute  ;  Thomas  Dwight,  Harvard 
University;  Simon  H.  Gage,  Cornell  University;  G.  Carl  Huber,  Michigan  University; 
George  H.  Huntington,  Columbia  University  ;  Franklin  P.  Mall,  Johns  Hopkins  University  ; 
J.  Playfair  McMurrich,  University  of  Toronto;  Charles  S.  Minot,  Harvard  University; 
George  A.  Piersol,  University  of  Pennsylvania.  Henry  McE.  Knower,  Secretary,  Johns 
Hopkins  University.  There  are  also  over  80  collaborators  connected  with  different 
institutions. 

The  American  journal  of  medical  research.     Boston,  1901+. 

The  American  journal  of  physiology.     Boston,  isc/ 

The  American  journal  of  microscopy  and  popular  science.      Illust.     New  York,  1876-1881. 

The  American  journal  of  science.     New  Haven,  1818     . 

The  American  monthly  microscopical  journal.     Illust.     Washington,  D.  C.,  1880— . 

American  Naturalist.  A  popular  illustrated  magazine  of  natural  history.  Salem  and 
Philadelphia,  Boston  and  New  York,  1867  . 

American  quarterly  microscopical  journal,  containing  the  transactions  of  the  New  York 
microscopical  society.  Illust.  New  York,  i8;S  . 

American  microscopical  society,  Proceedings.     1878-1894  ;  Transactions,  1895+. 

The  Anatomical  Record.  Baltimore,  1906—.  The  scope  includes  the  results  of  original 
investigations,  preliminary  reports,  reviews,  critical  notes,  courses  of  study,  laboratory 
plans  and  events,  including  appointments,  technique.  It  will  also  contain  the  Proceedings 
of  the  Association  of  American  Anatomists.  The  first  volume  of  the  Anatomical  Record 


*NHTK — When  a  periodical  is  no  longer  published,  the  dates  of  the  first  and  last  volumes 
are  given  ;  but  if  still  being  published,  the  date  of  the  first  volume  is  followed  by  a  plus  sign. 

See  Vol.  XVI  of  the  index  Catalog  of  the  Library  of  the  Surgeon  General's  office  for  a 
full  list  of  periodicals.  See  also  the  later  volumes  for  additions.  ' 

Besides  the  above-named  periodicals,  articles  on  the  microscope  or  the  application  of  the 
microscope  appear  occasionally  in  nearly  all  of  the  scientific  journals.  One  is  likely  to  get 
references  to  these  articles  through  the  Jour.  Roy.  Micr.  Soc.  or  the  Xeit.  wiss.  Mikroskopie. 
Excellent  articles  on  Photo-micrography  occur  in  the  special  Journals  and  Annuals  of 
Photography. 


344  niliLlOGRAPH  } ' 

was  published  with  the  American  Journal  of  Anatomy  (Nov.,  1906  to  Feb.,  1908)  and  under 
the  supervision  of  the  same  editorial  board.  It  is  now  independent,  with  the  following 
editors  :  Irving  Hardesty,  University  of  California  :  G.  Carl  Huber,  University  of  Michigan; 
Clarence  M.  Jackson,  University  of  Missouri  ;  Horace  Jayne,  the  Wistar  Institute  :  Thomas 
G.  Lee,  University  of  Minnesota  ;  Frederick  T.  Lewis,  Harvard  University  ;  Warren  H. 
Lewis,  Johns  Hopkins  University  :  Florence  R.  Sabin,  Johns  Hopkins  University  ;  George  I,. 
Streeter,  University  of  Michigan.  Published  by  the  Wistar  Institute  of  Anatomy  and 
Biology.  Philadelphia,  1908  —  . 

Anatomischer  Anzeiger.  Centrablatt  fur  die  gesammte  wissenchaftliche  Anatomic. 
Amtliches  Organ  der  anatomischen  Gesellschaft.  Herausgegeben  von  Dr.  Karl  Bardeleben. 
Jena,  1886  — .  Besides  articles  relating  to  the  microscope  or  histology,  a  full  record  of  current 
anatomical  literature  is  given. 

Annales  de  la  societe  beige  de  microscopic.     Bruxelles,  1874-. 

Archives  d'Anatomie  microscopique.     Illust.     Paris,  1897.     (Balbiani  et  Ranvier.) 

Archiv  ftir  miroscopische  Anatomic.    Illust.     Bonn,  1865    . 

Bibliographic  Anatomique.  Paris,  1893--- 

Centrablatt  fur  Physiologic.  Unter  Mitwirkung  der  physiologischen  Gesellschaft  zu 
Berlin,  Heraugsgegeben  von  S.  F.xner  und  J.  Gad.  Leipzig  and  Wien.  1887---.  Brief  extracts 
of  papers  having  a  physiological  bearing.  Full  bibliography  of  current  literature. 

English  mechanic.  London,  1866+.  Contains  many  of  the  papers  of  Mr.  Nelson  on 
lighting,  photo-micrography,  etc. 

Index  medicus.     New  York,  1879-  .     Bibliography,  including  histology  and  microscopy. 

International  journal  of  microscopy  and  popular  science.     London,  1890 -•-. 

Journal  of  anatomy  and  physiology.     Illust.     London  and  Cambridge,  1867-  . 

Journal  of  Applied  Microscopy  and  Laboratory  methods.     Illust.     Rochester,  X.  V  ,1^9^- . 

Journal  de  micrographie.    Illust.     Paris,  1877-1892. 

Journal  of  microscopy  and  natural  science.    London,  1885+. 

Journal  of  the  New  York  microscopical  society.     Illust.     New  York,  1885—. 

Journal  of  physiology.     Illust.     London  and  Cambridge,  18784. 

Journal  of  the  American  chemical  society.    New  York,  1879+. 

Journal  of  the  Royal  Microscopical  Society.  Illust.  London,  1878  +  .  Bibliography 
of  works  and  papers  relating  to  the  microscope,  microscopical  methods  and  histology.  It 
also  includes  a  summary  of  many  of  the  papers. 

Journal  of  the  Quekett  microscopical  club.    London,  186--    . 

The  Journal  of  Comparative  Neurology  and  Psychology.  This  Journal  was  founded  in 
1891  by  Clarence  L.  Herrick,  and  was  then  called  the  Journal  of  Comparative  Neurology. 
Since  1900  it  has  had  the  present  name.  The  editorial  board  consists  of  :  Henry  H.  Donald- 
son, C.  Judson  Herrick,  Herbert  S.  Jennings,  J.  B.  Johnston,  Adolph  Meyer,  Oliver  S.  Strong, 
John  B.  Watson,  Robert  M.  Yerkes.  It  is  now  published  by  the  Wistar  Institute  of  Anatomy 
and  Biology.  Philadelphia,  1908. 

The  Journal  of  Experimental  Zoology.  1904-  .  Editors:  William  K.  Brooks,  William 
E.  Castle,  Edwin  G.  Conklin,  Charles  B.  Davenport,  Herbert  S.  Jennings,  Frank  R.  Lillie, 
Jacques  Loeb,  Thomas  H.  Morgan,  George  H.  Parker,  Charles  O.  Whitman,  Edmund  B. 
Wilson.  It  is  published  by  the  Wistar  Institute  of  Anatomy  and  Biology,  1908. 

Journal  of  Morphology,  1887-1901.  1903,  1908-1  .  The  present  editors  are  Edward  Phelps 
Allis,  Jr.,  Edwin  G.  Conklin,  Henry  H.  Donaldson,  Milton  J.  Greenman,  Ross  G.  Harrison, 
G.  Carl  Huber,  Horace  Jayne,  Frank  R.  LilHe,  Franklin  P.  Mall,  Charles  S.  Minot,  Thomas 
H.  Morgan,  George  H.  Parker,  Charles  O.  Whitman,  Edmund  B.  Wilson.  The  Journal  of 
Morphology  is  now  published  by  the  Wistar  Institute  of  Anatomy  and  Biology.  Philadel- 
phia, 1908. 

The  Lens,  a  quarterly  journal  of  microscopy,  and  the  allied  natural  sciences,  with  the 
transactions  of  the  state  microscopical  society  of  Illinois.  Chicago,  1872-1873. 

The  Metallograpist,  a  quarterly  publication  devoted  to  the  study  of  metals  with  special 
reference  to  their  physics,  microstructure,  their  industrial  treatment  and  application.     Illus- 
trated especially  by  photo-micrographs  of  metals  and  alloys.     Boston,  1898+. 
The  Microscope.     Illust.     Washington,  D.  C.,  1881-1897. 


Bin  L I  OCR  A  PI  I Y  345 

Microscopical  bulletin  and  science  news.  Illust.  Philadelphia,  1883  •  .  The  editor, 
Edward  Pennock  introduced  the  term  "par-focal"  for  oculars  (see  vol.  iii,  p.  31). 

Monthly  microscopical  journal.     Illust.     London,  1869-1877. 

Nature-.     Illust.     London,  1869  —  . 

The  Observer.     Portland,  Conn.,   1890-1897. 

Philosophical  Transactions  of  the  Royal  Society  of  London.     Illust.     London,  1665     . 

Proceedings  of  the  American  microscopical  society,  1878     . 

Proceedings  of  the  Royal  Society.     London,  i1- 

(Ju.irterly  journal  of  microscopical  science.     Illust.     London,  1853  +  . 

Rev.  de  Metallurgie.     Paris,  1904     . 

Science,  a  weekly  journal  devoted  to  the  advancement  of  science.  New  York.  N.  S. 
1*95  •• 

Science  Record.     Boston,  1883-4. 

The  Scientific  American.     New  York,  18454  . 

X.eitschrift  f.  Angewandte    Mikroskopie.    1898+. 

/eitsehrift  ftlr  Instrumentenkuude.     Berlin,'  1881-  . 

Zeitschrift  ftir  physiologische  Chemie.    Strassburg,  1877    . 

X.eitschrift  fur  wissenschaftliche  Mikroskopie  und  fur  mikroskopische  Technik.  Illust. 
Braunschweig,  1884-.  Methods,  bibliography  and  original  papers. 


INDEX 


Abbe  apertometer 187 

Abbe  camera  lucida 142-152,  329 

Abbe  condenser  or  illuminator..  54-58 

Abbe  test-plate 185-187 

4 Aberration,  chromatic 4,  5,  185 

Cover-glass 64 

Spherical , 4,  5,  185 

Absorption  spectra 158-160,  167-172  . 

Acetylene  light 42,  60,  226 

Achromatic,  condenser 49,  50,  229 

Objectives 12,  15,  75 

Oculars  26 

Achromatism 15 

Actinic  focus 223 

Adjustable  objectives 14,  16,  64-68 

Experiments  with 64 

Micrometry 138 

Photo-micrography 235 

Adjusting  collar 65 

Adjustment,  of  analyzer 173 

Coarse  or  rapid,  and  fine 74 

Frontispiece  ;  of  objective_i4, 16,65 

Objective  for  cover-glass 65 

Aerial   image 35,37 

Air  bubbles 103-106 

Albumen  fixative,   Mayer's 271 

Alcohol,  absolute 271 

Ethyl 271 

Denatured 271 

Methyl 271 

Picric 281,  285 

Alcoholic  dye 311 

Amici  prism 155 

Amplifier 123 

Amplification  of  microscope 116 

Analyzer 163,  173 

Angle,  of  aperture 19,  20 

Critical     64 

Angstrom  and  Stokes'  law 160 

Angular  aperture 19,  20 

Anisotropic 175 

Apertometer,  Abbe's 187 

Aperture  of  objective 19,  23,  187 

Illuminating  cone 52 

Numerical  of  condenser 52 

Aplanatic   cone 54 

Objectives 15 

Ocular 15 

Apochromatic  condenser 50 

Objectives 15,  76,  221 


Apparatus  and  material, 

i,  39>  99,  n6,  141,  155,  185,  203,  220 

Appearances,  interpretation 99-115 

Arrangement,  minute  objects 261 

Serial  sections 318 

Tissue  for  sections 317 

Artifacts ico 

Artificial  illumination, 

42,  56,  60,  223,  228,  233 

Axial  light 4r 

Experiments  with 48 

Abbe  illuminator 56 

Axial,  point 19 

Ray   41 

Axis,  optic 2,  3,  12 

Crystals ---175 

Illuminator 57 

Secondary 5,  6,  57 


Back-ground   for    photographing 

" 207,  213 

Back   combination  or  system   of 

objective 1 2-14 

Bacterial  cultures,  photographing,  243 

Balsam 272 

Acid 272.  281,  315 

Bottle 257 

Mounting  in 257,  311 

Removal  from  lenses_: 71 

Natural 272 

Neutral  272 

Removal  from  slides 246 

Xylene 272 

Bands,  absorption 158 

Base  of  microscope,  Frontispiece, 

Bath,  water i 153 

Bibliography,  i,  38,  114,  139-140,  154, 
172,  184,  192,  202,  243,  261,  264,- 
283,  323,326,332,  333-345- 

Binocular 112-114 

Black,  anilin  for  tables 282 

Blocks,  for  shell  vials 259 

Blood,  absorption  spectrum  of 168 

Blotting  paper  for  models 326 

Board,  reagent 259,  268 

Body  of  microscope.  Frontispiece. 

Borax,  carmin 273 

Bottle    for    balsam,     glycerin    or 

shellac 257 

Reagent 271 


INDEX 


347 


Box,  glass 249 

Brownian  movement 109,  115 

Bubble,  air 103-106 

Bull's-eye 60,  224 

Engraving  glass 225 

Burning  point 7,  35 


Cabinet  for  microscopic    prepara- 
tions   264-265 

Calipers,  micrometer  or  pocket 249 

Camera,  bed 205 

Drawing 154 

Kmbryos 209 

Large,  transparent  sections 212 

Photo-micrographic 219,  222 

Testing 220 

Vertical j 205,  208,  219,  222 

Camera  lucida 141 

Abbe 142-152 

Wollaston's I2r,  124,  143,  144 

Canada  balsam 272 

Mounting  in 257,  311 

Removal  from  lenses 71 

Removal  from  slides 246 

Carbol-turpentine 274 

Carbol-xylene 274 

Carbon  monoxide  hemaglobin, 

spectrum  of 169 

Card,  catalog 264 

Centering 254 

Care  of,  eyes 72 

Microscope,  mechanical  parts..  70 

Optical  parts 69-72 

Negatives 207 

Water  immersion  objectives 68 

Carmin,  borax 273 

Mucus 273 

To  show  currents  and  pedesis, 

108,  109 

Spectrum 170 

Castor-x)'lene   clarifier 273 

Cataloging,  formula 262 

Preparations 261-1-64 

Cedar-wood  oil,  bottle  for 257 

Clearing 273 

Oil  immersion  objectives 273 

Cells,  deep,  thin 253 

Isolated  preparation 260 

Mounting 253 

Staining 260 

Cement,  shellac 253,  281 

Cementing  collodion 275,  302 

Center,  optical 2,  3 

Centering,  arrangement  of  illum- 
inator      --5o,  54 

Card 254 

Image  of  source  of  illuminarion  51 


Centimeter  rule u8 

Central  light 41,  48,  104 

Chamber,  moist 356 

Chemical  focus 15 

Microscope iyg 

Rays 15 

Scales 269 

Chemistry,  Micro 176 

Chloral  hematoxylin 278 

Chromatic,  aberration 4-5 

Correction 15,  185 

Objective 14 

Circle,  Ramsden 37 

Clarifier,  castor-xylene 273-274 

Class  demonstrations  in  histology 

and  embryology 193-202 

Cleaning,  back  lens  of  objective,  __  72 

Homogeneous  objectives 69 

Mixture  for  glass 250 

Optical  parts 70-72 

Slides  and  cover-glasses 246-249 

Water  immersion  objectives 68 

Clearer 273,  274,  311 

Clearing,  mixture 274 

Tissues L 311,  313 

Cedar-wood  oil 273 

Clinical  microscope 193 

Cloudiness,  of  objective  and  ocu- 
lar, how  to  determine 101 

Removal 71 

Coarse  adjustment  of  microscope, 

Frontispiece  ;    testing 74 

Cob-web  micrometer 133 

Collective  lens 28 

Collodion 274 

Coating  glass  rod 107 

Cementing 275,303 

Clarifying 309 

Fastening  sections  to  slide 275 

Hardening 305 

Method 304-310 

Collodionizing  sections 302 

Color,  correct  photography 214 

Correction 15 

Images 64,  68 

Law  of 160 

Production  of 176 

Screens 214-217,  244 

Colored,  minerals  spectra  of 171 

Comparison  prism 163,  164 

Spectrum 164,  169 

Compensation   ocular 28,  29 

Complementary  spectra 160 

Compound  microscope,  see  under 
microscope. 

Concave  lenses  3 

Mirror,  use  of 42-43 

Condenser _ 48-58 


348 


INDEX 


Abbe 54-58 

Achromatic 49,  50,  229 

Apochromatic 50 

Bull's  eye 60,  224 

Centering 50,  54 

Illuminating  cone 52 

Mirror  with 55 

Non-achromatic 55 

Numerical  aperture 52 

Optic  axis 50,  54 

Photo-micrography 224,  229 

Standard  size 30,  55 

Substage 49 

See  also  illuminator. 

Condensing  lens 40 

Cone,  aplanatic 54 

Illuminating 52 

Congo-glycerin 275 

Red  275 

Conjugate  foci   4 

Construction  of  images,  geometri- 

cal 5 

Continuous,  spectrum 158 

Contoured,  doubly 107 

Converging  lens 3 

Lens  system n 

Convex  lenses 3-6 

Correction,    chromatic,    or     color 

4,  15,  185 

Cover-glass 65,  66 

Over  and  under  correction 15 

Cotton,  collodion,  gun  or  soluble. 274 

Counterstaining 310-317 

Cover-glass,  or  covering  glass 248 

Aberration  by 64 

Adjustment,  specific  directions  65 
Adjustment  for,  in  photo-micro- 
graphy   235 

Adjustment     and     tube-length 

16,  17,  65 

Anchoring 255 

Cleaning 248-249 

Correction. 64,65 

Effect  on  rays  from  object 21,  65 

Gauges 249-250 

Measurer 249,  250 

Measuring  thickness  of 249 

Non-adjustable  objectives, table 

of  thickness 18 

No.  I,  variation  of  thickness 250 

Putting  on 103,  252 

Sealing  ___ 254 

Serial  sections 322 

Thickness 17,  18,  249,  322 

Tube-length 174 

Wiping 248 

Critical  angle 64 

Crystals  from  frog  for  pedesis.  ._  ..no 
Systems 180 


Crystallization   under    microscope 

58,    177-180 

Crystallography 176 

List  of  substances 179-180 

Currents,  diffusion,  avoidance 313 

Liquids 108 

Cutting  sections 289 


D 


Dark-ground   illumination. 42,   56-60, 
198 

Abbe  illuminator 58 

Mirror 57 

Dark  room  for  drawing 153 

Daylight,  lighting 40 

Decalcifier 275 

Deck-plugs  for  collodion  blocks 305 

Dehydration 258,  271,  311 

Demonstration ,  microscopes.  _  1 92 -i 94 

Micro-projection  apparatus  200-201 

Denatured  alcohol 271 

Deparaffining 302 

Designation  of  oculars 29 

Wavelength 165 

Determination   of    field  of  micro- 
scope   33 

Equivalent  focus 190-192 

Magnification 116,  191 

Working  distance 47 

Diamond,  writing 319 

Diaphragms  and  their  employment 

42,  43,  50-53 

Diffusion  currents,  avoidance 313 

Direct,  light 40 

Vision  spectroscope 155 

Disc,  Ramsden 37 

Dissecting  microscope 10,  194 

Dissociating  liquids 275 

Dissociator,  formaldehyde 275 

Miiller's  fluid 275 

Nitric  acid 261,  275,  280 

Distance,  principal  focal 4,  7,  35 

Standard  at  which  the  virtual 
image  is  measured 123 

Working   d.    of  simple   micro- 
scope or  objective 47 

Working  d. of  compound  micro- 
scope  13,  39,  47 

Distinctness  of  outline 105 

Distortion  in  drawing,  avoidance.  143 

Diverging  lens 3 

Double  spectrum 164 

Vision 116,  118 

Doubly  contoured 107 

Refracting  175 

Draw-tube,  Frontispiece. 

Pushing  in 44 


INDEX 


349 


Drawing,    with   Abbe   camera   lu- 

cida M6-I53 

Board  for  Abbe  camera  lucida, 

146-149.  329 

Distortion,    avoidance 143 

Embryograph  for 154 

Microscope 140 

Models 329 

Photographic  camera 154 

Photo-engraving 324 

Room    for    projection     micro- 
scope  153, 154 

Scale  and  enlargement 151 

With  simple  microscope 153 

Drugs,  adulteration 182 

Dry  objectives 14,  20-23 

Light  utilized 21 

Dry  mounting 252 

Numerical  aperture 20 

Dry  plates,  discovery  by  Mad- 
clox . 218 

Dust,   of  living   rcoms,  examina- 
tion  in 

On  objectives  and  oculars,  how 

to  determine 101 

Removal 71 

Dye,  general  staining  with 310 

Aqueous . 311 

Alcoholic 311 

E 

Eccentric  diaphragm 5r,  57,  58 

Egg  pipette 319 

Eikonometer 137,  138 

Elastic  stain 275,  315 

Embryograph 154 

Embryos,  camera   for 209 

Photographing 209-212 

Records 211 

Serial  Sections 320 

Engraving  glass  for  condenser 225 

Enlargements 242 

Eosin 276 

Equivalent  focal  length  or  focus  of 

objectives  and  oculars__i3,  29, 

33,   190-192 

Erect  image i 

Erecting,     binocular     microscope 

112-113 

Etching  for  metallography 241 

Ether,  alcohol 276 

Sulfuric 276 

Ethylalcohol 271 

Examination  of  dust  of  living 
rooms,  bread  crumbs,  corn 
starch,  fibres  of  cotton,  lin- 
en, silk,  human  and  animal 
hairs,  potatoes,  rice,  scales 


of    butterflies    and     moths, 
wheat in 

|  Experiments,  Abbe  condenser 56 

Adjustable  and   immersion   ob- 
jectives   64 

Compound  microscope 30 

Homogeneous  immersion  objec- 
tive    68 

Lighting  and  focusing 42,  43 

Micro-chemistry 176, 181 

Micro-spectroscope 167 

Micro-polariscope 174 

Mounting 251 

Photo-micrography 226 

Simple  microscope 6 

Exposure,  of  photographic  plates. 

226,  233,  236,  242 

Color-screen 214-217,  244 

Extraordinary    ray     of     polarized 

light 173 

Eye  and  microscope i,  7,  9,  11,36 

Eyes,  care  of 72 

Emmetropic,    hyperopic,     my- 
opic, normal 8,  9 

Muscae  volitantes no 

Eye-lens  of  the  ocular 25 

Eye- piece 25 

Micrometer 129 

Parfocal 45 

Eye-point .7,  37,  130,  142 

Ocular,   demonstration 37 

Eye-shade,  adjusting 73 


Farrants'  solution 276 

Fibers,  examination HI 

Textile 181 

Field 32 

Camera  lucida 121 

Illumination 52,  61 

Orthoscopic  ocular 27 

Periscopic  ocular 27 

View  with  Microscope_32-34,  118, 

142-145 

Size  of,   with   different  objec- 
tives and  oculars 33,  34 

Field-lens,  of  ocular 25-27 

Action *_ 37 

Dust  on 101 

Filar,  micrometer  ocular 26,  30 

Ocular  micrometer I33~I35 

Filtering  balsam 272 

Fine    adjustment,     Frontispiece  ; 

Testing 74 

Fir,  balsam  of 272 

Fixation _. 284 

Fixative,  albumen,  Mayer's 271 

Fixer 284 


350 


INDEX 


Fluid,   Miiller's 275,  280,  283 

Fluid,  Zenkers' 283 

Fluorite  lens 15 

Focal  distance,  or  point,  principal 

4,  7,  35 

Length  equivalent „    13 

Focus 6 

Actinic 223 

Chemical 15 

Conjugate 4,  6 

Equivalent,   of   objectives  and 

oculars 13,  29,  190 

Principal 4,  5,  7 

Virtual 3 

Visual 15,  223 

Focusing, 7,  39 

Adjustments,  testing 74 

Compound  microscope 39 

Experiments 43 

Glass 207,  210 

High  objectives 45 

Low  objectives 43 

Objective     for     micro-spectro- 
scope   166 

Photo-micrography 226 

Screen   for   photo-micrography 

210,    226 

Simple  microscope 7,  39 

Slit  of  micro-spectroscope 167 

Food,  detection  of  adulteration 182 

Form  of  objects,  determination 103 

Formal 260 

Formalin.-   276 

Formaldehyde 276 

Dissociator 275 

Isolation 260 

Percentages 270 

Formula,  for  aperture 20 

Cataloging 262 

Desired   percentages 270 

Equivalent  focus 191 

Refraction 62 

Fraunhofer  lines 158-159 

Free,  hand  sections 289 

Working  distance 39 

Front  combination  or  lens  of  ob- 
jective   12-14 

Frontal  sections * 321 

'Fuchsin,  acid 281 

Basic 275 

Picro 281 

Function  of  objective 34~35 

Ocular 36 


Gauge,  cover-glass 250 

Gauze,  absorbent 246 

Gelatin,  liquid 279 


Geometrical  construction  of  images     5 

Glass,  cleaning  mixture 251 

Ground 34,  210,  225 

Rod  appearance   under   micro- 
scope  106,  107 

Slides  or  slips 245 

Glasses,  graduate 269 

Watch 260 

Glue,  liquid,  preparation  of 279 

Glycerin 277 

Congo 275 

Mounting  objects 255 

Glycerin  jelly  for 277 

Anatomic  preparations 277 

Microscopic  preparations 277 

Mounting  objects 255 

Glycogen,  iodin-  stain 278 

Graduates 269 

Greenoagh's  binocular  microscope 

112,  113 

Ground  glass,  focusing  screen. 210,  226 

Preparation 34 

Gun  cotton --274 

H 

Half-tones 324 

Hardening  collodion 305 

Tissue 284 

Hematein 278 

Hematoxylin,  chloral 278 

Hemoglobin   spectrum 159 

High  school  microscope 75 

Histology,  physiologic 264 

1  History  of  photo-micrography 217 

Holder,  lens 10,124,319 

Slide . 302,  312 

Homogenous  immersion,   conden- 
ser   54 

Objective 14,  19-23 

Cleaning 69 

Experiments 68 

Numerical  aperture I9~23 

Homogenous  liquid 14 

Tester 68,  188 

Hones  and  honing 288 

Horizontal  camera 233 

Huygenian  ocular 26,  27,  37,  130 


Illuminating,  cone,  aperture 52 

Objective 16,  184 

Power 24 

Illumination 48 

Abbe  camera  lucida 150 

Artificial 42,  56,  60 

Air  and  oil 103-106 

Centering  image 51 


INDEX 


351 


Dark  ground 42,  56-60,198 

Daylight 40 

Entire  field 61 

Lamp 61 

Methods    39-64 

Micro-polariscope 174 

Micro-spectroscope 165 

( )blique  with  air  and  oil 104 

Opaque  objects 166,  239 

Photography 226 

Photo-micrography 232 

\Yall aston's  camera  lucida 144 

Illuminator . 48-58 

Vertical 16,  184,240 

See  also  condenser. 

Image,  aerial 35,  37 

Color 64,  68 

Flame 52 

( jeometrical  construction 5 

Inverted,  real  of  objective 35 

Object, size  and  position, 5,  9,  ii,  122 

Real 5,  9,  u,  12,  35-37,  116 

Refraction 61,  68 

Retinal 7,  u,  36 

Swaying  of 56 

Virtual 5,  7,  9,  n,  123 

Image-power  of  objectives 22 

Imbedding 293,  305 

Immersion,  fluid  or  liquid. 14,  68/273 

Illuminator 54 

Objective 14,  20,  68-69 

Incandescence  or  line  spectra 158 

Incident  light 40 

Index,    medium    in   front   of  ob- 
jective  20-23 

Refraction 62 

Indicator  ocular 197 

Infiltration,  collodion 304 

Paraffin 291 

Paraffin  dish . 292 

Ink  for  labels,  catalogs,  drawing_324 

Interpolation 20 

Interpretation  of  appearances  un- 
der the  microscope 99-115 

lodin  stain  for  glycogen 278 

Iris  diaphragm 181 

Irrigating  with  reagents 255 

Isochromatic  plates 214 

Isolation 259 

Formaldehyde 260 

Nitric  acid 261 

Isostigmar  objective 206 

Iso  tropic 174 

J-K 

Japanese  filter  or  tissue  paper 70 

Jar  for  slides,  etc.  1 246-249 

Jelly,  glycerin 277 


Jena  glass 12 

Jurisprudence,  micrometry 140 

Knife,  sharpening 286-289 

Support 299 


Labels  and  catalogs 262 

Labeling    microscopical     prepara- 
tions  262 

Photographic  negative 207 

Serial  sections ___323 

Laboratory  compound  microscope  74 
Table  73 

Lamp,  acetylene 42,  60,  226 

Alcohol  or  spirit 301 

Black 279 

Condenser 153,  330 

Electric 42,  226 

Petroleum 42,  60,  226,  233 

Lantern 200 

Slides 242 

Law  of  color 160 

Lens,  concave 3 

Converging 3 

Convex  3-6 

Eye 25 

Field -25-27 

Fluorite__i 15 

Holder 10,  124,  319 

Paper 70 

System n 

Thick 3 

Letters,  in  stairs 102 

Photo-engraving _ 324 

Lettering  oculars 29 

Light,  with  Abbe  illuminator 56 

Acetylene... 42,  60,  226 

Artificial . 42,  56,  60,  226 

Axial 41,  48,  56 

Direct 40 

Central 41,  48,  104 

Electric 42,  226 

Incident 40 

Mirror 42,  43 

Oblique 41,  48,  56 

Petroleum 42,  60,  226,  233 

Photo-micrography 226 

Polarized 173 

Reflected   40 

Sun _• 226 

Transmitted 41 

Utilized   with   different    objec- 
tives    21 

Vertical  illuminator 240 

Wave  length  of 164 

Welsbach 42,  226 

Lighting 39-80 


352 


INDEX 


Abbe  camera  lucida 150 

Artificial 42,  56,  60,  232 

Experiments 42 

Horizontal  camera 233 

Micro-polariscope 174 

Micro-spectroscope 165 

Mirror 43,  48 

Daylight 40,  226 

Photography 210,  213 

Photomicrography 228 

Vertical  illuminator 240 

See  illumination 

Line  spectrum 158-159 

Liquid,  currents  in 108 

Homogeneous 14,68,  273 

Locker,  Laboratory  or  Student 268 

Longisection 317 

M 

Magnification,  of  compensation  oc- 
ulars   28 

Effect  of  adjusting  objective__i38 

Determination 16-140,  191 

Eikonometer 137 

Expressed  in  diameters 116 

Initial  or  independent 191 

Microscope 116 

Microscope  with  Abbe  camera 

lucida 151 

Microscope,  compound 119 

Microscope,   simple 117 

Photo-micrographs 232 

Real  images 116 

Table 126 

Projection  microscope 117,  330 

Varying  with  compound  micro- 
scope   123 

Velocity 108 

Magnifier,  tripod 9,  117,  207 

Marker  for  preparations 80 

Marking  objects 80,  199 

Negatives 213,  238 

Objectives 32 

Masks  for  preparations 201 

Measure,  metric,  cover  2nd  p 

118,140 

Unit  of,  in  Micrometry 127 

Wave   length 165 

Measurer,   cover-glass 250 

Measuring  thickness  of  cover-glass 

249 

Measurement  with  microscope  and 

micrometer,  Ch.  IV. 
Mechanical  parts  of  compound  mi- 
croscope, Frontispiece.il,  74-75 

Care  70 

Testing 73 

Mechanical  stage Fig.  76-78 


Mercuric  chlorid 279 

Crystals 279,  283,  315,  317,  320 

Metallic  surfaces,  photography 239 

Preparation   241 

Metallography,  microscope 183 

Metals,  examination 183,  239 

Met-hemaglobin, spectrum  157, 169,244 

Method,  collodion 304-310 

Paraffin 291-304 

Methyl  alcohol 271 

Methylated  spirits  271 

Methylene  blue ._. 280 

Metric  measures  and  equivalents, 

cover  2nd  p. 118 

Micro-chemistry 176-181 

Slides 1 79 

Micro-metallography,  objects 183 

Micrometer 116 

Calipers 249 

Cob-web 133 

Combined  ocular 135 

Filar  m.  ocular 133,   134 

Filling  lines 119 

Net 147 

Ocular  and  stage 139 

Objector  objective 119 

Ocular  or  eye-piece 129-138 

.Ocular,  micrometry_ 131 

Ocular,  ratio 132 

Ocular,  valuation 130,   135,   136 

Ocular,  varying  valuation 136 

Photo-micrography 232 

Screw   ocular 133 

Stage 119 

Table  of  magnification 126 

Micrometry  126-140 

Adjustable  objectives 138 

Comparison  of  methods 139 

Compound  microscope 127 

Eikonometer 137 

Jurisprudence 140 

Limit  of  accuracy  in 139 

Ocular  micrometer 131 

Simple  microscope 126 

Remarks  on 138 

Unit  of  measure  in 127 

Micro-millimeter 127 

Micron 127 

Measuring  wave  length  of  Iight_i6s 

Micro-photograph 217 

Micro-polariscope 163,  173-176 

Micro-polarizer 173 

Micro-projection 153,  200 

Drawing 153,  330 

Magnification 117 

Masks  for  specimens 201 

Microscope,  care 70 

Amplification 116 


INDEX 


353 


Binocular  ._. i 12-1 14 

Chemical 178 

Clinical 193 

Demonstration 192-193 

Dissecting 10,  194 

Erecting 1 12-1 13 

Field 32-34,  142-145 

Focusing 39 

Illumination  for,  Ch.  II. 

Magnification 116 

Metallography 183 

Micro-chemical  analysis 176 

Opaque  objects    -.238 

Photo -micrography 227 

Polarizing 173 

Preparation,       with       erecting 

prism 113 

Projection 153,  154,  200-201 

Price 75 

Putting  an  object  under 31 

Screen 69 

Stand  for  large,  transparent  ob- 
jects  212 

Stand,  for  embryos 209 

Solar 200 

Traveling 195,  196 

Traversing 182 

Microscope  compound II 

Drawing 140 

Figures,  Frontispiecei2,  82-98,  1 13, 
178,  182,  193, 195,  198,  202,  227,  230 

Focusing 39 

High  schools 74 

Laboratory- 74,  82-98 

Lamp 60,  61 

Magnification 116-126 

Magnification  of  drawing  with 

Abbe  camera  lucida 151 

Mechanical  parts ir,  74 

Micrometry 127 

Optic  axis 12-14 

Optical  parts ir,  74 

Quality  and  cost 75 

Testing . _ 73 

Varying  magnification 123 

Working  distance 13,  40,  47 

Micro-summar 210 

Microscope,  simple i 

Drawing 153 

Experiments 6 

Figures 9,  10,  124,  192,  194,  207 

Focusing 7,  39 

Images 7 

Magnification  __ 137,  117,124 

Micrometry 126 

Obtaining  focus 7 

Working  distance .. 39 

Microscopic,  objective ,,„,„,  n 


Objects,   drawing 141 

Ocular 25 

Slides  or  slips 245 

Tube-length 17-19,  66 

Vision 25 

Microscopic  preparations,   cabinet 

264-265 

Cataloging 262-264 

Labeling 261-264 

Mounting 251-261 

Trays 266 

Micro-spectroscope 155-172 

Adjusting 160 

Direct  vision    .    155 

Experiments 167 

Focusing  the  slit 161 

Lighting 165 

Objectives  to  use 166 

Reversal  of  colors 155 

Slit,  mechanism 156,  160 

Micro  Tessar  objective 209 

Microtomes 286 

Figures  -290, 295, 296,  297,  306,  307 

Micrum * 127 

Mikron 127 

Milk  globules,  to  overcome  pedsis.ns 

Minerals,  absorption  spectra 171 

Minot's  microtome 295 

Minute  objects,   arrangement 261 

Mirror 12-14 

Abbe   illuminator 55 

Arrangement  for  drawing 146 

Concave,  use  of: 43 

Dark  ground  illumination 56-60 

Light  with,  central  and  oblique  48 

Lighting, 40 

Plane,  use  of_  43 

Mixture,  clearing 274 

Cleaning 250 

Models : 324 

Blotting  paper 326 

Drawing , 329 

Wax 325 

Moist,  chamber 256 

Molecular  movement 109 

Monazite  sand,  spectrum 171 

Mounting  251,  311 

Cells,  preparation 253-258 

Low   powers 241 

Media  and  preparation 252 

Objects  for  polariscope 174-176 

Permanent 252 

Temporary 251 

Mounting  objects 251 

Balsam 257,272,  311 

Dry  in  air 252 

Glycerin 255 

Glycerin  jelly 255,  257 


354 


INDEX 


Media  miscible  with  water 254 

Minute  objects 261 

Opaque  objects 241 

Permanent 252 

Resinous  media,   by  drying  or 

desiccation 257-258,  311 

Resinous  media,   by  successive 

displacements 258,  311 

Temporary 251 

Movement,  Brownian,  or   molecu- 
lar   109 

Mucicarmin  or  mucus  stain 

273,  315-317 

Miiller's  fluid 280,  283 

Dissociator 275 

Muscae  volitantes 1 10 

Muscular  fibers,  isolation 261 

Polarizing  object 175 

Museum  jar 246-247 

N 

Natural  balsam 272 

Negative,  labeling 207,238 

Oculars '. 25 

Record 238 

Storing 207 

Net  micrometer. 147 

Neutral  balsam 272 

Red r 280 

Nicol  prism 173 

Nitric  acid 280 

Dissociator 275 

Nomenclature  of  objectives 13 

Non-achromatic  condenser 54 

Objectives 14 

Non-adjustable  objectives. 16,  18 

Normal  liquid 280 

Salt  or  saline  solution 275,  280 

Nose-piece 31+46 

Marking  objectives 32 

Thread  or  screw-thread 77 

Numerical  apeiture,  of  condenser.  52 

Objectives 19,  23,  187 

Table 23 

o 

Object,  determination  of  form 103 

Image,  si/.e  of 12,  122 

Marking  parts 8o-(- 

Marking  position 197,  199 

Micrometer 119 

Mounting 251,  311 

Putting  under  microscope 31 

Shading 69 

Suitable  for  photo-micrography  225 
Transparent   with   curved   out- 
lines,   relative    position    in 
microscopic  preparations 103 


Objective 1 1-25 

Achromatic 12,  15 

Adjustable 14,  16,  64-66 

Adjustable,  micrometry 138 

Adjustable,  photo-micr 235 

Adjustment 64 

Aerial  image 35 

Aperture '. 19,  20,  187 

Aplanatic 15 

Apochromatic 15,  221 

Back  combination 13,  14 

Cleaning 70-72 

Collar,    graduated   for    adjust- 
ment  65 

Cloudiness  or  dust,  how  to  de- 
termine   101 

Designation 13 

Dry 14,  20-24 

Equivalent  focus 13,  33,  190 

Field  . 33,  34 

Focusing      for     micro-spectro- 
scope    166 

Front  combination 13,  14 

Function 34~35 

Glass  for 12,  15 

High,  focusing 45 

Homogeneous   immersion.    ... 

14,  19-23,^8 

Homogeneous    immersion, 

cleaning   69 

Homogeneous   immersion,    ex- 
periments   68 

Illuminating 16,  184,  239 

Image,  power 22 

Immersion  14,  20,68 

Index  of  refraction  of  medium 

iti  front 21,  23 

Initial  magnification 191 

Inverted,  real  image 35 

Isostigmar 206 

Laboratory  microscope 75 

Lettering 13 

Light  utilized 21 

Low,  focusing 43 

Magnification 191 

Marking,  bv  Krauss'  method.  _  32 

Micrometallography   16,183 

Micro-polariscope 174 

Microscopic -   ii 

Microtessar 209 

Micro-spectroscope 166 

Nomenclature 13 

Non-achromatic 14 

Non-adjustable 16 

Non-adjustable,  table 18 

Nose-piece 31,  32,  46,83+ 

Numbering 13 

Numerical  aperture 19,  23,  187 


INDI'.X 


355 


Oil  immersion 14 

Pantachromatic 16 

Para-chroinatic 16 

Par-focal . .  3*1,46 

Photography  .. 206-210 

Photo-micrography 221 

Projection  ... 16 

Putting  in  position  and  remov- 
ing   30 

Screw-thread 77 

Section  16 

Semi-apochromatic 16 

Table  of  field 33 

Terminology 13 

t'nadjustable 16 

Variable   16 

Visual  and  actinic  foci 223 

Water  immersion 19-23,  66 

Working  distance 13,  39,  40,  47 

Oblique  light,   with  Abbe  illumi- 
nator      56 

Mirror 48,  57 

Ocular,  various  forms 25-28 

Aplanatic 15 

Cleaning 70-72 

Cloudiness,  how  to  determine 

and   remove 71,101 

Compensation 28,  29 

Equivalent  focus 29,  33,  191 

Eye-point 26,  37 

Field-lens 37 

Filar     or     screw      micrometer 

30,   133-136 

Function 36 

Huygenian 26,  27,  37,  130 

Indicator 80,  197 

Iris  diaphragm 181 

Lettering  and  numbering 29 

Micrometer,  micrometry__i29-i36 

Negative 25 

Parfocal 27,  43 

Photo-micrography 221 

Pointer ' So,  197 

Positive 26 

Power 29 

Projection 29,  223 

Searching 28 

Spectroscopic _.  155 

Standard  size 30 

Table "_,  26 

Working 28 

Oil,  and  air  appearances  and  dis- 
tinguishing optically 103-106 

Cedar-wood 273 

Oil-globules,     with     central     and 

oblique  illuminations 104 

Oil  immersion  objectives 14 

Opaque  objects,  lighting 183,  238 


Photography 238 

Optic  axis 2,3,  12 

Condenser  or  illuminator 54 

Crystals 175 

Microscope 12   14 

Optical i 

Center 2 

Focus 15 

Parts  of  compound  microscope 

Frontispiece  and n,   74 

Section 108 

Order  of   procedure   in   mounting 

objects  dry  or  in  air 252 

Glycerin  and  glycerin  jelly 255 

Resinous  media  by  desiccation. 257 
Resinous  media   by    successive 

displacement 258,  311 

Ordinary  ray,  with  polarizer 173 

Orthochromatic   plates 214 

Orthoscopic  ocular,  field 32 

Outline  distinctness 105 

Oven  paraffin 293 

Over-correction..: 5 

Oxy-hemoglobin,  spectrum__i59,  169 


Paper,  bibulous,  filter,  lens,  or  Jap- 
anese  for   cleaning   oculars 

and  objectives 70 

Blotting  for  models 326 

Paraffin 280 

Filtering 281 

Infiltrating 292 

Imbedding 293 

Method 291-304 

Oven 293 

Removing  from  sections 302 

Wax _^ 280 

Parfocal  objectives 47 

Oculars 1 43-45 

Pedesis 109,    no 

Overcoming 115 

Polarizing  microscope no 

Penetrating  power 24 

Pentration  of 'objective 24 

Percentages,  of  liquids 270 

Permanent,  mounting 252 

Preparation  of  isolated  cells 260 

Permanganate  of  Potash,  spectrum 

157,  168 

Petri  dish -249 

Photographing    bacterial    cul- 
tures  243 

Petroleum  light 42,60,   226,   233 

Pharmacological  products,  exami- 
nation  182 

Photo-engraving,  drawing  and  let- 
tering  324 


356 


INDEX 


Photographic,  camera 203-205 

Negatives 207-213,  238 

Objectives 206,  209,  210 

Prints 207 

Photography  of  bacterial  cultures. 243 

Color-correct 214 

Colored  objects 214 

Compared     with    photo-micro- 
graphy   217-220 

Embryos 209 

Focusing  and  exposure 

204-206,210 

Indebtedness    to    photo-micro- 
graphy  217 

Large  transparent  objects_2i2-2i4 

Lighting 206,213,  239 

Metallic   objects 239 

Objectives 206,  209,  210 

Objects  in  alcohol  or  water 204 

Opaque  objects 238,  241 

Plates 2T4 

Stage 208 

Vertical  camera 204-210 

Photo-micrograph 217 

Determination  of  magnification 

232 

5-20  diameters 209 

20-50  diameters .228 

100-2000  diameters 232-236 

Metallic   surfaces, 238-242 

Objects   suitable 225 

Opaque  objects 238-242 

Prints 207 

Plates 214 

Reproductions 234 

With  and  without  an  ocular 

229-237 

Photo-micrographic,  camera 

219-222,  233 

Outfit : 220 

Stand 227,  230 

Photo-micrography 203-244 

Apparatus 220,  233 

Compared   with  ordinary   pho- 
tography  215,  220 

Condenser 49,  224,  229 

Distinguished  from  nricro-pho- 

'  tography 217-220 

Cover-glass  correction 235 

Experiments 226 

Exposure. 211,  214,  217,231,236,242 

Focusing __2o6,  210,  226 

Focusing  screen 206 

Lighting 

210,  223-226,  229,  232,  239,  240,  243 

Micrometer  formagnification__232 

Objectives  and  oculars 

16,  221,  237-240 


Staining  preparations 215 

Vertical  camera 208,  219,  222 

With  and  without  ocular__229-237 

Record  table 238 

Physiologic  histology .'_.  264 

Picric-alcohol. 281 

Picro-fuchsin 281,  314 

Pillar  of  microscope,  Frontispiece. 

Pin-hole  diaphragm 54 

Pjpette 300,  311 

Egg- 319 

Plane  mirror,  use 43 

Plates,  color-correct 214 

Isochromatic    or  orthochro- 

matic 214 

Pleochroistn 175 

Pleurosigma  angulatum 48 

Point,  axial 19 

Burning 7,  35 

Eye 37,  130,  142 

Pointer  ocular 80,  197,  176 

Polarized  light,  extraordinary  and 

ordinary  ray 173 

Polarizer  and  analyzer 162,  173 

Polarizing  microscope,  pedesis 

no,    175 

Position  of  condenser 54 

Objects  or  partsof  same  object.  102 

Positive  oculars 12,  26 

Power,  of  microscope 116 

Illuminating,    penetrating,    re- 
solving, of  objective 24-25 

Ocular 29 

Preparation  of  reagents 268-283 

Preparations,  cataloging 261 

Cabinet 264 

Labeling 262 

Permanent 252 

Temporary 251 

Principal,  focus 3,  4.  7 

Focal  distances 4,  35 

Optic  axis 2,  7,  12 

Prism  of  Abbe  camera  lucida. 144-148 

Amici 155 

Comparison 164 

Dispersing 158 

Erecting •__.  1 13 

Nicol 173 

Slit  of  micro-spectroscope,  mut- 
ual arrangement 161 

Wollaston's  camera  lucida.  143-144 

Prints,  photographic 207 

Projection,  microscope 

153,  154, 200-201,  330 

Objective 16 

Ocular 29,  223 

Pyroxylin 274 


INDEX 


357 


Q-R 

yuandrant  for  camera  Iucida_i46,  148 

Ratnsden  circle  or  disc 37 

Ratio,  ocular  micrometer 132 

Razor  and  support 299 

Reagent 268-283 

Board 268 

Bottle 271 

Real  image 6,  9,  n,  12,  35-37,  116 

Record,  of  embryos 211 

Negatives 213,238 

Record  table,  collodion  method  -.310 

Negatives 238 

Paraffin  method 303 

Red,  congo 275 

Neutral 280 

Reflected  light 40 

Reflection,  total 64 

Refraction,  images 61,  68 

Index 62 

Medium  in  front  of  objective 

20-23 

Refractive,  doubly 175 

Highly 107 

Singly  175 

Relative   position   of   microscopic 

objects 103 

Resinous  media, mounting  objects, 257 
Resolution   and    numerical    aper- 
ture   24 

Resolving  power 24 

Retinal  image 7,  9,  n,  12 

Revolving  nose-piece 31,  32 

Ribbon  sections 296 

Deparaffining 302 

Electrification 298 

Spreading 298 

Storing 298 

Tray 266,  268 


Sagittal  sections 322 

Salicylic  acid,  crystallization 58 

Salt  solution,   normal 280 

Scale,  of  drawing 151 

Size  of  photographs 204 

Wave  lengths 164 

Scales,  chemical 269 

Screen,  color 215-217 

Focusing  for  photography, 206,  210 

Ground  glass 34 

Microscope 69 

Screw,  society 76 

Micrometer 30,  133-135 

Sealing  cover-glass 254,  256 

Searching  ocular 28 

Secondary  axis 367 


Section  knife  and  sharpening 

287-289 

Lifter 309 

Optical 108 

Sections,  arrangement  of  tissue 294 

Clearing 274.  311 

Cutting 289-323 

Dehydration '_.  311 

Deparaffining 302 

Extending  with  water 298 

Fastening  to  slide 298-303,  308 

Free  hand 289 

Freezing 290 

Frontal 321 

Longi- 317 

Mounting 311 

Ribbon 295 

Sagittal 322 

Serial 317 

Spreading  or  stretching  by  heat 

. 298 

Staining ; 310 

vSurface 318 

Trans- 317,   321 

Transferring 308 

Vertical 318 

Selenite  plate  for  polariscope 176 

Semi-apochromatic  objective 16 

Serial  sections 317-323 

Embryos 320-323 

Sharpening  section   knives 288 

Shell  vials 259,  285 

Shellac  cement 253,  281 

Significance  of  aperture 23 

Silvering 281 

Simple  microscope,  see  under  mi- 
croscope. 
Sines,  table  of,  3d  page  of  cover. 

Slides 245 

Cleaning 245-247 

Holder 302 

Micro-chemistry 179,  245 

Tray 266-268 

Sliding  microtome 306-307 

Slips,  glass 245 

£lit  mechanism  of  micro-spectro- 
scope  156,  160 

Society  screw 76 

Sodium,  lines  and  spectrum 

157,  158,  244 

Solar  microscope 200 

Spectrum  or  s.  of  sunlight. 

157,  158,  244 

Soluble  cotton 274 

Solution,    Farrants' 276 

Percentage 270 

Saturated 269 

Spectral,  colors 158 


358 


INDEX 


Ocular I55i  !6o 

Spectroscope 155 

Direct  vision —  155,  167 

Spectroscopic,      examination      of 

color-screens 216 

Ocular 155 

Spectrum 157-172,  244 

Absorption 158,  159,  160,  167 

Analysis 172 

Angstrom  and  Stokes'  law 160 

Banded 170 

Blood 1 68 

Carbon  monoxide  hemogloblin_  169 

Carmin  solution 170 

Colorless  bodies 171 

Color  screens 244 

Comparison . 164 

Complementary __i6o 

Continuous 158 

Double 164 

Incandescence 158 

Line _: 158 

Met-hemoglobin 157,  244 

Minerals,  monazite  sand 171 

Oxy-hemoglobin 159,  169 

Permanganate  of  potash 

. 157,  168,  244 

Single-banded    of   hemoglobin 

159,  169 

Sodium 157,  158,  244 

Solar 157,  158,  244 

Two-banded    of    oxy-hemoglo- 

bin 169 

Spherical  aberration 4,  5 

Test  .   185 

Stage,  Frontispiece,  mechanical__  82 

Stain,  alcoholic  and  aqueous 311 

Counter 310-317 

Elastic 315,  275 

Staining 260,  310-317 

Stand,  microscope 75 

Photo-micrographic 98,  227 

Special  for  embryos 208 

Special    for   large    transparent 

objects 212 

Standard,   distance    (250  mm.)    at 
which   the  virtual  image  is 

,         measured 123 

Screw 77-8o 

vSize  for  condenser 30,  55 

Size  for  oculars 30 

Starch,    determination   by   polari- 

scope 175 

Stender  dish 260,  305 

Stokes  and  Angstrom's  law  of  ab- 
sorption spectra  160 

Strops  and  stropping 289 

Storing  negatives 207 

Preparations 264 


Ribbons  of  sections 298 

Student  locker __268 

Substage,  Frontispiece. 

Substances  for  crystallography 

179-180 

Sudan  III 282 

Sulphonal  with  polarizer 176 

Sulphuric  or  sulfuric  ether 276 

Support  for  knife  of  microtome___299 

Surface  sections 318 

Swaying  of  image 56 

System,  back,  front,  intermediate 

of  lenses 12-14 

Crystal 180 

Metric,  cover  2nd  p __i4o 


Table,  black 282 

Collodion  method 310 

Immersion  fluid 189 

Laboratory 73 

Magnification  and  valuation  of 

ocular  micrometer 126 

Oculars 26 

Tube-length   and   thickness  of 

cover-glasses 18 

Natural   sines,    third    page    of 
cover. 

Numerical  aperture 23 

Paraffin  method 303 

Record,  photography 238 

Size  of  fields 33 

Testing  homogeneous  liquids.- 189 
Valuations  of  ocular  microme- 
ter  126 

Weights  and  measures,  2d  page 
of  cover. 

Temporary  mounting 251 

Terminology  of  objectives 13 

Test   of   chromatic   and   spherical 

aberration 185-188 

Tester,  cover-glass 250 

Homogeneous  liquids 68,  188 

Testing  a  camera 220 

A  microscope  and  its  parts 73 

Test-plate,    Abbe's,      method     of 

using 185-187 

Textile  fibers,  examination__iu,  181 
Thickness,  of  cover-glass  for  non- 
adjustable  objectives 18 

Serial  sections 321 

Thread,   standard    for    nose-piece 

and  objective 77 

Tissues,  arranging  for  sections 294 

Fixing  or  hardening 284-286 

Washing  apparatus 286 

Transections 317,  321 

Transferring  sections 308 


INDEX 


359 


Transmitted  light 41 

Traversing  microscope 182 

Tray  for  ribbons  or  slides' 266-268 

Triplet,  Hastings 10 

Tripod 9,  117 

Focusing  glass 207 

Tube  of  microscope,  Frontispiece. 

Tube-length 17-19 

Cover-glass  adjustment 66-67 

Importance 66 

Various  opticians,  table 18 

Turn-table 253 

u 

I  aratnicroscopy • 59 

Unadjustable  objectives 16 

ruder-correction     5 

Tnit  of  measures,  in  micrometry.  127 
Wave  length 165 


Valuation  of  ocular  micrometer 

-_- 126,  130,  135,  136 

Variable   objective.. 16 

Varying    magnification    of     com- 
pound  microscope 123 

Varying  ocular  micrometer  valua- 
tion  136 

Velocity  under  microscope 108 

Vertical,  camera 203,  222 

Illuminator 16,   184,  239 

Sections 318 

Vials,  preparation  and  shell 

259,   284-285 

Blocks 259,  268 

Virtual  image 7,  9,  11,  12,  36 

Standard    distance     at     which 

measured 123 

Visibility  with  objectives 24 


Vision,  double 116,  118 

Microscopic 25 

W 

Washing  apparatus  for  tissues 286 

Waste  bowl 309 

Watch  glass- 260 

Water  immersion  objective 19-23,  66 

Light    utilized 21 

Numerical  aperture 22,  23 

Water,   bath 153 

Wave  length,  designation 165 

Scale   164 

Wax,  bees 325 

Models 325 

Paraffin 280 

Weigert's  elastic  stain 275,  315 

Weights  and  measures,  see  2d  page 
of  cover. 

Welsbach  light 226 

Wenham's  binocular  microscope 

'. 112,    1 14 

Wollaston's  camera  lucida  

121,  124,  143,  144 

Work-room  for  photo-micrography  221 

Work-table,  position,  etc. ..   73 

Working,  distance   of   microscope 

or  objective 13,  39,  40,  47 

Ocular 28 

Writing  diamond 3 1 9 

X 

Xylene 273 

Balsam 272 

Xylol,  German  form  of  xylene 273 


Zenker's  fluid 283 


185862 

Gage,,  S.  H. 

QH211 

The  micro 

scope* 

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LIBRARY,  BRANCH  OF  THE  COLLEGE  OF  AGRICULTURE 


UNIVERSITY    OF    CALIFORNIA 
3RANCH    OF    THE    COLLEGE    OF    AGRICULTURE 

THIS  BOOK  IS  DUE  ON  THE  LAST  DATE 
STAMPED  BELOW 


TABLK  OF  NATURAL  SINES 
Compiled  from  Prof.  G.  W.  Jones'  Logarithmic  Tables 


MINUTES. 

DEGREES  AND 

QUARTER  DEGREES  UP  TO 

90°. 

i  '0.00029 

i° 

0.01745  16°, 

0.2756431°, 

0.5150446°, 

o.7i934i6i°, 

0.87462 

76°, 

0.97030 

2  0.00058 

i°,  15'  0.0218  1  16°,  15  '0.27983  31°,  15 

'0.5  1877  46°,  15 

'  0.72236  6i°,'i5'  0.87673  76°,  IS'Q.  97134 

3  0.00087 

1,30 

0.02618  16,30 

0.2840231,30 

0.5225046,30 

0.7253761,30 

0.87882  76,30 

0.97237 

4  0.00116 

i,45 

0.03054  16,45 

0.2882031,45 

0.52621  46,45 

0.7283761,45 

0.8808976,45 

0.97338 

5  0.00145 

2 

0.0349017 

0.2923732 

0.5299247 

0.7313562 

0.88295  77 

0-97437 

6  0.00175 

2,15 

0.0392617,15 

0.2965432,15 

0.53361  47,i5 

0.7343262,15 

0.8849977,15 

0-97534 

7  0.00204 

2,30 

0.04362  17,30 

0.30071  32,30 

0-5373047,30 

0.7372862,30 

0.88701  77,30 

0.97630 

8  0.00233 

2,45 

0.04798  17,45 

0.30486  32,45 

0.54097  47,45 

0.74022  62,45 

0.88902  77,45 

0.97723 

9  0.00262    3 

0.05234  1  8 

0.30902  33 

0.5446448 

0.74314  63 

0.89101  78 

0.97815 

100.00291    3,15 

0.05669  18,15 

0-3131633,15 

0.5482948,15 

0.7460663,15 

0.89298  78,15 

0.97905 

ii  0.00320 

3,30 

0.06105  18,30 

0.3173033,30 

0.5519448,30 

0.7489663,30 

0.89493  78,30 

0.97992 

12  0.00349 

3,45 

0.0654018,45 

0.3214433,45 

0-55557  48,45 

0.75184  63,45 

0.89687  78,45 

0.98079 

13  0.00378 

4 

0.06976  19 

0.3255734 

o.559r949 

0.75471  64 

0.89879  79 

0.98163 

14  0.00407 

4,15 

0.07411  19,15 

0.3296934,15 

0.5628049,15 

0.7575664,15 

0.90070 

79,15 

0.98245 

15  0.00436 

4,30 

0.07846  19,30 

0.33381  34,30 

0.56641  49,30 

0.76041  64,30 

0.90259  79,30 

0.98325 

16  0.00465 

4,45 

0.08281  19,45 

0.33792  34,45 

0.57000  49,45 

0.7632364,45 

0.90446  79,45 

0.98404 

17  0.00495 

5 

0.08716  20 

0.34202  35 

0.57358  50 

0.76604  65 

0.90631  80 

0.98481 

1  8  0.00524 

5,15 

0.09150  20,15 

0.3461235,15 

0.5771550,15 

0.7688465,15 

0.90814  80,15 

0.98556 

19  0.00553 

5,30 

0.09585  20,30 

0.35021:35,30 

0.58070  50,30 

0.77162  65,30 

0.90996 

80,30 

0.98629 

20  0.00582 

5,45 

0.1001920,45 

0.35429  35,45 

0.58425  50,45 

0.7743965,45 

0.91176 

8o,45 

0.98700 

21  O.Oo6ll 

6 

O.I04532I 

0-35837  36 

0.5877951 

0.7771566 

0.91355 

8l 

0.98769 

22  0.0064O 

6,15 

0.1088721,15 

0.3624436,15 

0-59131  5i,i5 

0.77988  66,15 

0.91531 

81,15 

0.98836 

23  0.00669 

6,30 

O.II320  2I,3O 

0.3665036,30 

0.5948251,30 

0.78261  66,30 

0.91706 

81,30 

0.98902 

24  0.00698 

6,45 

0.1175421,45 

0.3705636,45 

0.5983251,45 

0.78532  66,45 

0.91879 

8i,45 

0.98965 

25  0.00727 

7 

O.I2I87  22 

0.3746137 

0.60182  52 

0.78801  67 

0.92050 

82 

0.99027 

26  0.00756 

7,i5 

0.1262022,15 

0.3786537,15 

0.6052952,15 

0.7906967,15 

0.92220 

82,15 

0.99087 

27  0.00785 

7,30 

0.1305322,30 

0.3826837,30 

0.60876  52,30 

0.7933567,30 

0.92388 

82,30 

0.99144 

28  O.OO8l4 

7.45 

0.13485  22,45 

0.38671  37,45 

0.61222  52,45 

0.7960067,45 

0.92554 

82,45 

0.99200 

29  O.OO844 

8 

0.1391723 

0.3907338 

0.6156653       • 

0.79864  68 

0.92718 

83 

0.99255 

30  0.00873 

8,15 

0.1434923,15 

o.39474|38,i5 

0.6190953,15 

0.80125:68,15 

0.92881 

83,15 

0.99307 

31  O.OO9O2 

8,30 

0.1478123,30 

0.39875  38,30 

0.62251  53,30 

0.8038668,30 

0.93042 

83,30 

0-99357 

32  0.00931    8,45 

0.1521223,45 

0.4027538,45 

0.6259253,45 

0.80644  68,45 

0.93201 

83,45 

0.99406 

33  0.00960   9 

0.1564324 

0.40674  39 

0.62932154 

0.80902  69 

0.93358 

83 

0.99452 

340.00989   9,15 

0.16074  24,15 

0.4107239,15 

0.63271  54,15 

0.8115769,15 

0.935M 

84,15 

0.99497 

35  0.01018   9,30 

0.16505  24,30 

0.4146939,30 

0.63608  54,30 

0.81412  69,30 

0.93667 

84,30 

0-99540 

36  0.01047   9,45 

0.1693524,45 

0.4186639,45 

0.6394454,45 

0.81664  69,45 

0.93819 

84,45 

0.99580 

37  0.01076  10 

0.1736525 

0.42262  40 

0.6427955 

0.81915  70 

0.93969 

84 

0.99619 

38  0.01105  10,15 

0.1779425,15 

0.4265740,15 

0.6461255,15 

0.82165  70,15 

0.94118 

85,15 

0.99657 

39  0.01134  10,30 

0.18224  25,30 

0.43051  40,30 

0-64945  55,30 

0.8241370,30 

0.94264 

85,30 

0.99692 

40  0.01164  10,45 

0.1865225,45 

0.4344540,45 

0.6527655,45 

0.82653  70,45 

0.94409 

85,45 

0.99725 

41  0.01193  IJ 

0.19081  26 

0.43837)41 

0.65606:56 

0.82904  71 

0.94552 

85 

0.99756 

42  0.01222  11,15 

0.1950926,15 

0.44229  41,15 

0.65935156,15 

0.8314771,15 

0.94693 

86,15 

0.99786 

43  0.01251  11,30 

0.19937  26,30 

0.44620  41,30 

0.66262  56,30 

0.8338971,30 

0.94832 

86,30 

0.99813 

44  0.01280  1  1,45 

0.2036426,45 

0.4501041,45 

0.6658856,45 

0.83629  71,45 

0.94970 

86,45 

0.99839 

45  0.01309  12 

0.20791  27 

0-45399  42 

0.66913  57 

0.8386772 

0.95106 

86 

0.99863 

46  0.01338  12,15 

0.2121827,15 

0.45787142,15 

0.6723757,15 

0.84104  72,15 

0.95240 

87,15 

0.99885 

47  0.01367  12,30 

0.21644  27,30 

0.4617542,30 

0-67559  57,30 

0.8433972,30 

0.95372 

87,30 

0.99905 

48  0.01396  12,45 

0.22070  27,45 

0.46561  42,45 

0.6788057,45 

0.84573  72,45 

0.95502 

8/,45 

0.99923 

49  0.01425  13 

0.22495  28 

0.4694743 

0.68200  58 

0.84805  73 

0.95630 

87 

0-99939 

500.0145413,15 

0.22920  28,15 

0.4733243,15 

0.6851858,15 

0.8503573,15 

0-95757 

88,15 

0-99953 

51  0.01483  13,30 

0.2334528,30 

0.47716  43,30 

0.68835  58,30 

0.85264  73,30 

0.95882 

88,30 

0.99966 

52  0.01513  13,45 

0.2376928,45 

0.4809943,45 

0.6915158,45 

0.85491  73,45 

0.96005 

88,45 

0.99976 

53  0.01542  14 

0.24192  29 

0.48481144 

0.69466,59 

0.8571774 

0.96126 

88 

0.99985 

54  0.01571  14,15 

0.2461529,15 

0.48862  44,15 

0.69779  59,15 

0.85941  74,15 

0.96246 

89,15 

0.99991 

55  0.01600  14,30 

0.2503829,30 

0.49242  44,30 

0.70091  59,30 

0.86163  74,30 

0.96363 

89,30 

0.99996 

56  0.01629  14,45 

0.2546029,45 

0.4962244,45 

0.70401  59,45 

0.86384  74,45 

0.96479 

89,45 

0.99999 

57  0.01658  15 

0.2588230 

0.5000045 

0.70711  60 

0.8660375 

0-96593 

90 

I.OOOOO 

580.01687  15,15 

0.2630330,15 

0.5037745,15 

0.71019  60,15 

0.8682075,15 

0.96705 

. 

590.01716  15,30 

0.26724  30,30 

0.5075445,30 

0.7132560,30 

0.87236  75,30 

0.96815 

. 

. 

600.01745  15,45 

0.2714430,45 

0.5112945,45 

0.7163060,45 

0.87250  75,45 

0.96923 

•     • 

